Human PAK65

ABSTRACT

A novel human serine protein kinase, human p21-protein activated serine kinase p65 protein, referred to as hPAK65, and methods for its preparation and use are provided. Nucleic acids encoding hPAK65 and methods for their use in preparing hPAK65 as well as in preparing and identifying hPAK65 analogs are provided. Methods provided for the use of hPAK65 protein and its protein fragments, such as those that retain at least one hPAK65 activity, that include screening libraries of agents for candidates that modulate hPAK65 activity. Methods are provided to identify agents that modulate the interaction of hPAK65 with rho-like p21 GTPases, particularly rac 1 and CDC42Hs binding to hPAK65 and subsequent activation of hPAK65 serine protein kinase activity, that modulate hPAK65 serine protein kinase activity, and that modulate hPAK65 effect on p21 protein GTPase activity. Such modulating agents can provide novel chemotherapeutic agents for treatment of neoplasia, lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases, apoptosis, and the like, that are related to hPAK65 and p21 protein signal transduction pathways.

This application is a continuation of U.S. application Ser. No.08/780,833, filed Jan. 10, 1997, now U.S. Pat. No. 5,698,428, which is acontinuation of U.S. application Ser. No. 08/475,682, filed Jun. 7,1995, now U.S. Pat. No. 5,605,825, which is a continuation of U.S.application Ser. No. 08/369,780, filed Jan. 6, 1995, now U.S. Pat. No.5,518,911.

INTRODUCTION

1. Technical Field

This invention relates to methods for making and using and compositionscontaining human p21-activated kinase p65 ("hPAK65") nucleic acid orprotein sequences.

2. Background

The rho-like proteins (i.e., p21 proteins), like other GTPases, cyclebetween an active GTP-bound form and an inactive GDP-bound state (Nobesand Hall, 1994). Regulation of these forms was shown to be controlled byseveral proteins including guanine nucleotide exchange factors ("GEF")such as Dbl and GTPase activating proteins (Hart et al. 1991, Boguskiand McCormicK, 1993). Members of the rho family of proteins, includingRhoA, B, C, rac 1, 2, CDC42Hs ("CDC42 Homo sapien"), and TC10, share atleast 50% sequence identity with each other and 30% identity with otherras-like proteins (Nobes and Hall, 1994). Insight into the physiologicalfunction of rho and rac proteins emerged from recent reports describedby Ridley and Hall (Ridley and Hall, 1992; Ridley et al., 1992), inwhich rapid cytoskeletal effects were detected when rho and rac proteinswere micro injected into Swiss 3T3 fibroblasts. Activated rho inducesstress fiber. formation and focal contact (Ridley and Hall 1992) whereasactivated rac induces the formation of membrane ruffles and lamelipodia(Ridley et al. 1992). Rho proteins are also implicated in otherphysiological roles associated with cytoskeletal rearrangements such ascell motility (Takaishi et al. 1993), cytokinesis (Kishi et al. 1993)and lymphocyte aggregation (Tominaga et al 1993).

Although the physiological function of CDC42 was shown to be essentialin bud formation in yeast (Johnson and Pringle 1990), no similarphysiological function was described for its mammalian homologueCDC42Hs. However, a hint for its role in mammalian cells came from astudy demonstrating that the protooncogene Dbl exhibits GEF activity onrho and CDC42Hs (Hart et al. 1991). This observation suggests a role forrho-like proteins in cell transformation to a neoplatic state. However,not all proteins containing the Dbl domain demonstrate nucleotideexchange activity on rho-like proteins. For example, vav (Gulbins et al.1993), Ect2 (Miki et al. 1993), ras GRF and bcr (Boguski and McCormick1993) do not exert nucleotide exchange activity on rho, and the Dbldomain of bcr and ras GRF do not transform cells. A recent studysuggests a direct link for rho and Dbl in vivo by demonstrating that vavand Dbl transformation is mediated by rho (Khosravi-Far et al. 1994).

p21 proteins are known to be integral components of signal transductionmechanisms leading to control of cell proliferation. Many pathologicalconditions result, at least in part, from aberrant control of cellproliferation or differentiation. For example, neoplasia ischaracterized by a clonally derived cell population which has adiminished capacity for responding to normal cell proliferation controlsignals. Oncogenic transformation of cells leads to a number of changesin cellular metabolism, physiology, and morphology. One characteristicalteration of oncogenically transformed cells is a loss ofresponsiveness to constraints on cell proliferation and differentiationnormally imposed by the appropriate expression of cell growth regulatorygenes.

Currently only a few effector molecules for rho-like proteins are knownincluding rat brain kinase PAK65 (Manser et al. 1994) and p67-phox ofNADPH oxidase (Diekmann et al 1994). From molecular function studies inphagocytes, it was demonstrated that rac 1 and 2 are involved in thecontrol of superoxide generation by the NADPH oxidase (Abo et al. 1991,Knaus et al. 1991). Activated rac together with two other oxidasecytosolic components, p47-phox and p67-phox, assemble with the membranebound cytochrome b₅₅₈ to form an active oxidase (Segal and Abo 1993).The effector molecule for rac in this system is p67 phox (Diekmann etal. 1994). An additional molecular effector for rac and CDC42Hs wasshown to be a rat brain serine/threonine kinase, which is activated byrac 1 and CDC42Hs (Manser et al. 1994). Other studies suggested thatCDC42Hs and rho also can activate P13 kinase (Zhang et al. 1993; Zhenget al. 1994).

In view of the potential and varied roles for rho-like p21 proteins inphysiological pathways and disease states, such as cell structuralintegrity, physiological roles associated with cytoskeletalrearrangements such as cell motility, cytokinesis, lymphocyteaggregation, tumor cell transformation and proliferation, metasteses,cell aggregation, and the paucity of understanding of the molecules andagents that selectively effect or modulate the activities of theseproteins in one or more of these physiological pathways, there thusexists a need in the art for compounds and agents with effector andmodulator activity and methods to identify these and relatedcompositions and agents. Further, such agents can serve as commercialresearch reagents for control of cell proliferation, differentiation,and other p21-related conditions. Despite progress in developing a moredefined model of the molecular mechanisms underlying the transformedphenotype and neoplasia, few significant therapeutic methods applicableto treating cancer beyond conventional chemotherapy have resulted. Suchp21 protein modulating agents can provide novel chemotherapeutic agentsfor treatment of neoplasia, lymphoproliferative conditions, arthritis,inflammation., autoimmune diseases, apoptosis, and the like. These andother objects are provided by this invention.

Relevant Literature

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2. Hart, M. J., Eva, A., Evans, T., Aaronson, S. A., Cerione, R. A.(1991) Nature 354, 311-314.

3. Boguski, M. S., McCormick, F. (1993) Nature, 366, 643-654.

4. Ridley, A. J., Hall, A. (1992) Cell 70, 389-399.

5. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., Hall,A. (1992) Cell 70, 401-410.

6. Takaishi, K., Kikuchi, A., Kuroda, S., Kotani, K., Sasaki, T., Takai,Y. (1993), Mol. Cell. Biol. 13, 72-79.

7. Kishi, K., Sosaki, T., Kuroda, S., Itah, T., Takai, Y. (1993) J.Cell. Biol. 120, 1187-95.

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SUMMARY OF THE INVENTION

The invention provides isolated polynucleotides comprising nucleic acidsequences encoding a novel human p21-protein activated serine kinase p65protein ("hPAK65"). The invention provides isolated polynucleotideshaving nucleic acid sequences encoding hPAK65, preferably as describedin FIG. 2A (SEQ ID NO: 1), nucleic acid sequences complementary to thatsequence, nucleic acid sequences containing degenerate codonreplacements within the human PAK65 coding sequence of that sequence,allelic variants of that sequence, closely related variants having atleast 95% homology to that sequence, and fragments at least 10 bases inlength from those sequences and which will selectively hybridize tonucleic acids encoding hPAK65. The nucleic acids sequences arepreferably those found in nature, although in view of this invention thepolynucleotides containing these sequence can be prepared in numerousways known in the art, including synthetic methods.

Also provided are hPAK65 recombinant constructs, e.g. fusions,truncations, substitutions, that provide polypeptides having certaindesirable properties such as constitutive serine kinase activity, p21protein binding activity, and ease of purification and identification.

Also provided are isolated and purified hPAK65 proteins containing thesequnces found in the polynuclotides of the invention. Methods forpreparation of hPAK65 proteins are provided, including isolation fromnatural sources, synthetic production, and recombinant production usingthe nucleic acid sequences provided by the invention. The inventionprovides a human hPAK65 protein, and fragments thereof, having an aminoacid sequence depicted in FIG. 2A.

The invention includes vectors and transformed host cells for expressingthe isolated polynucleotides of the invention when the isolatedpolynucleotides are operably linked to an expression vector appropriatefor expression in the host cell used.

The isolated proteins of the invention having serine protein kinaseactivity are used to generate phosphorylated proteins and amino acids.Peptides of the invention can also be used to generate antibodies fordetection assays and isolation methods of PAK65 and PAK65-complexes inPAK-65-related signal transduction pathways and disease conditions. Theisolated polynucleotides the invention can find further use in thedissection of and in particular antisense treatments for PAK65-relatedsignal transduction pathways and disease conditions.

The invention also provides compositions and methods to screen librariesof agents for their ability to modulate or inhibit the properties ofhPAK65, which as disclosed herein include its protein kinase activity,its p21-protein binding activity, its p21-protein inducedautophosphorylation activity, and its p21-bound phosphate releaseactivity. Preferably the agents modulate the rac1- andCDC42Hs-interacting properties of human PAK65. The invention providescompositions and methods for treating or preventing neoplasia in humanand veterinary patients, compositions and methods for screening alibrary of agents for pharmacological activity in regulating cellproliferation and/or cell differentiation, compositions and methods formodulation of a transformed cell phenotype in vitro, including use inbioprocess control and as commercial laboratory reagents. The presentinvention is also directed to pharmaceutical compositions for thecontrol of hPAK65-dependent diseases in mammals which includes an agentcapable of modulating at least one of the properties associated withhPAK65 and to a method of controlling hPAK65dependent diseases whichincludes administering to a mammal suffering from a hPAK65 kinasedependent disease a hPAK65 kinase dependent disease controlling amountof an agent capable of modulating one of the properties associated withhPAK65. Mammal has the usual meaning and includes humans. Pharmaceuticaluses are intended to include veterinary uses, especially use indomesticated animals such as cattle, sheep, pigs, goats, dogs, cats,rabbits, hamsters, gerbils, rats, and mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict fractionation of neutrophil cytosol. FIG. 1Bdepicts a chromatogram showing fractionation of neutrophil cytosol on aMono Q column, on which 10 ml (10 mg/ml) were applied and eluted with 30ml of salt gradient. The collected fractions were analyzed by an overlayassay with [γ³² P]GTP CDC42Hs as a probe (FIG. 1A) as described indetail in the EXAMPLES section.

FIGS. 2A, 2B, and 2C depict the nucleotide (SEQ ID NO: 1) and deducedamino acid sequence (SEQ ID NO: 2) of human PAK65 ("hPAK65"). hPAK65cDNA was cloned from human placenta library. FIG. 2a presents a nucleicacid sequence of human PAK65 cDNA and its deduced amino acid sequence.In FIG. 2A the underlined amino acids correspond to the peptide sequenceobtained from p65 purification. FIG. 2B depicts a comparison between thededuced amino acid sequences of hPAK65 and rat brain PAK65. FIG. 2Cdepicts a comparison between a putative kinase domain of hPAK65 andyeast STE20.

FIGS. 3A, B and C present Northern blot analysis of hPAK65 in tissue andcell lines. Radioactively labeled probe generated by PCR from the kinasedomain (nucleotide sequence 1009-1912) of hPAK65 cDNA was used tohybridize mRNA isolated from various tissues immobilized on northernblots. Autoradiograph was exposed 3 hrs.

FIG. 4 depicts hPAK65 binding specificity among rho-like proteins. 80 μgof neutrophil cytosol or 2-3 μg of recombinant hPAK65 were applied onSDS-PAGE, blotted onto a PVDF filter, and analyzed by the overlay assay.The filter was probed with the indicated GTPase preloaded with [γ³²P]GTP or [β³² P]GDP. Lanes a contain recombinant hPAK65 and lanes bcontain neutrophil cytosol. Protein molecular weight markers areindicated in kilodaltons.

FIGS. 5A-C present results of activation of hPAK65 autophosphorylation.In FIG. 5A 1-2 μg of recombinant hPAK65 immobilized on beads in 40 μlkinase buffer, were incubated with 1-2 μg of the indicated GTPase whichwere preloaded with either GTP or GDP. The reaction was incubated for 20min. at 30° C. with. 50 μM ATP and 5 μCi [γ³² P]ATP. Phosphorylatedproteins were analyzed on SDS PAGE followed by an autoradiography. InFIG. 5B 4 μg of radio labeled phosphorylated hPAK65 mediated by CDC42Hswere hydrolyzed in 6 N HCl, 110° C. for 2 hrs, and the phosphoaminoacids were separated on a thin layer electrophoresis. ³² Pi labeledresidues were detected by autoradiography. In FIG. 5C proteolyticdigestion was performed with the indicated enzymes (trypsin,chymotrypsin, endoproteinase Glu-C) on hPAK65 which was eitherpreincubated with rac1 or CDC42Hs in a kinase reaction containing [γ³²P]ATP. The radiolabelled peptides were resolved on a 16% Tricine gel andwere visualized by autoradiography.

FIGS. 6A-D depict activation of hPAK65 kinase activity. In FIG. 6A 3 μgof MBP were included with hPAK65 in a kinase reaction as described forFIG. 5A. In FIG. 6B 5 μl of the kinase reaction mixture was removedevery 5 min and the reaction was stopped by adding SDS sample buffer.The phosphorylated MBP was separated on a 14% SDS PAGE, the band wasexcised, and the incorporated ³² Pi was counted. In FIG. 6C 4 μg ofhPAK65 (30 μl beads) were first incubated for 20 min with 3 μg CDC42Hsin the presence (activated) or the absence (control) of ATP in a kinasereaction. To remove CDC42Hs, the hPAK65 beads were washed three timesand hPAK65 was subjected to a second kinase reaction containing [γ²P]ATP and MBP. FIG. 6D presents hPAK65 kinase activity as counts perminute incorporated into MBP isolated from the blots of FIG. 6A.

FIG. 7 presents a comparison of the binding of CDC42Hs to phosphorylatedand unphosphorylated hPAK65. 2 μg of either unphosphorylated orphosphorylated hPAK65 (induced by CDC42Hs) were run on a SDS PAGE,stained with Coomassie Blue, and were also tested for CDC42Hs binding bythe overlay assay. Lane a contains unphosphorylated form and lane bcontains the phophorylated form.

FIGS. 8A and 8B depict the effect of hPAK65 on intrinsic and p190stimulated GTPase activity of CDC42Hs. 1 nM CDC42Hs preloaded with [γ²P]GTP was incubated with the indicated concentrations ofunphosphorylated or phosphorylated hPAK65 in the absence (FIG. 8A) orthe presence (FIG. 8B) of 20 nM of the catalytic domain of p190,followed by the phosphate released assay.

FIG. 9 presents a schematic of a model of the role of rac1/CDC42Hs inthe activation of hPAK65 kinase. In step 1 rac1 or CDC42Hs exchangefactors, which are stimualted by growth factors, stimulate the releaseof GDP from rac1 or CDC42Hs and subsequently the binding of GTP to rac1or CDC42Hs. In step 2 the activated rac1 or CDC42Hs binds to hPAK65. Instep 3 rac1 or CDC42Hs induces the autophosphorylation of hPAK65 toactivate hPAK65 serine kinase activity. In step 4 the intrinsic GTPaseactivity of rac1 or CDC42Hs hydrolyses the GTP to the inactive GDPstate. In step 5 the GDP-bound form of rac1 or CDC42Hs dissociates fromthe active autophosphorylated hPAK65 kinase.

FIG. 10 presents the amino acid sequence of the p21-binding fusionprotein GST-PAKette.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Novel compositions comprising isolated polynucleotides andoligonucleotides having nucleic acid sequences of hPAK65 are provided bythe invention. Isolated proteins encoded by hPAK65 polynucleotides arealso provided by the invention. Exemplary nucleic acid and amino acidsequences are set forth in SEQUENCE ID NO's 1 and 2, respectively.Aspects of the invention include isolated polynucleotides andoligonucleotides having nucleic acid sequences homologous to SEQUENCE IDNO: 1 or encoding SEQUENCE ID NO:2; and isolated proteins having aminoacid sequences homologous to SEQUENCE ID NO: 2. Embodiments of theinvention are achieved by either chemically synthesizing polynucleotidesor oligonucleotides encoding hPAK65, in whole or in part, having atleast 80% nucleic acid sequence homology (with preferably increasinghomologies to 100%) to a human PAK65 nucleic acid sequence; or byisolating polynucleotides encoding a naturally occurring hPAK65 havingat least 80% nucleic acid sequence homology (with preferably increasinghomologies to 100%) to a nucleic acid sequence encoding human PAK65. Forinstance, techniques for synthesizing polynucleotides andoligonucleotides are well known in the art and changes can be made tothe sequence of SEQUENCE ID NO: 1 that allow for deviation from thatsequence while permitting at least a 80% nucleic acid sequence homologyto be maintained with the sequence described in SEQUENCE ID NO: 1. Inone preferred embodiment the sequences have at least 95% homology to thecoding sequence of SEQUENCE ID NO 1.

Alternatively, in another embodiment of the invention polynucleotidesencoding naturally occurring hPAK65 with at least 80% nucleic acidsequence homology to a human PAK65 nucleic acid sequence are isolatedusing isolated polynucleotides or oligonucleotides having nucleic acidsequences derived from SEQUENCE ID NO: 1. Hybridization and washconditions are known in the art, and discussed herein, that can be usedto selectively hybridize probe nucleic acids generated from the sequencedescribed in SEQUENCE ID NO: 1 to nucleic acids with at least 80%nucleic acid sequence homology to human PAK nucleic acid sequence.

In the isolated protein aspects of the invention, isolated proteins ofhPAK65 have amino acid sequences that correspond to a hPAK65 having atleast 80% amino acid homology (with increasing preference for sequenceswith at least 85%, 90%, 95%, 99%, to having one amino acid difference)to a human PAK65 sequence, preferably to that of SEQUENCE ID NO 2. Theisolated proteins of the invention can be expressed using thepolynucleotides of the invention operably linked to an appropriatecontrol sequence in an expression vector suitable for expression ineither a mammalian, insect, yeast, or bacterial cell.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the laboratory procedures in cell culture, moleculargenetics, and nucleic acid chemistry and hybridization described beloware those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, and microbial culture and transformation (e.g.,electroporation, lipofection). Generally enzymatic reactions andpurification steppe performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N. Y., which is incorporated herein by reference)which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, organicsynthetic chemistry, and pharmaceutical formulation described below arethose well known and commonly employed in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalformulation and delivery, and treatment of patients. As employedthroughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings:

The term "isolated polynucleotide" referred to herein means apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthere of, which by virtue of its origin the "isolated polynucleotide"(1) is not associated with all or a portion of a polynucleotide in whichthe "isolated polynucleotide" is found in nature, (2) is operably linkedto a polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term "isolated protein" referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin the "isolated protein" (1) is not associatedwith proteins found in nature, (2) is free of other proteins from thesame source, e.g. free of human proteins, (3) is expressed by a cellfrom a different species, or (4) does not occur in nature.

The term "polypeptide" is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred p21-interacting hPAK65 polypeptides include: the humanfull-length protein comprising the polypeptide sequence shown in FIG.2A, polypeptides comprising a kinase domain, such as consistingessentially of a sequence shown in FIG. 2C, polypeptides comprising ap21-binding domain consisting essentially of amino acids 49 to 113 ofFIG. 2A, polypeptides of hPAK65 missing amino acids 489, 490 and/or 491,or polypeptides of hPAK65 having amino acids corresponding to 380, 383and/or 384 of FIG. 2A replaced with Glu or Asp.

The term "naturally occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term "operably linked" referred to herein refers to a juxtapositionwherein the components so described are in a relationship permittingthem to function in their intended manner. A control sequence "operablylinked" to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences.

The term "control sequence" referred to herein refers to polynucleotidesequences which are necessary to effect the expression of codingsequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term "control sequences" is intended to include, at aminimum, all components whose presence is necessary for expression, andcan also include additional components whose presence is advantageous,for example, leader sequences and fusion partner sequences.

The term "polynucleotide" as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term "oligonucleotide" referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset with 200 bases or fewer inlength. Preferably oligonucleotides are 10 to 60 bases in length andmost preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases inlength. Oligonucleotides are usually single stranded, e.g. for probes;although oligonucleotides may be double stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides of the invention can beeither sense or antisense oligonucleotides. The term "naturallyoccurring nucleotides" referred to herein includes deoxyribonucleotidesand ribonucleotides. The term "modified nucleotides" referred to hereinincludes nucleotides with modified or substituted sugar groups and thelike. The term "oligonucleotide linkages" referred to herein includesoligonucleotides linkages such as phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phoshoraniladate, phosphoroamidate, and the like. An oligonucleotide caninclude a label for detection, if desired.

The term "sequence homology" referred to herein describes the proportionof base matches between two nucleic acid sequences or the proportionamino acid matches between two amino acid sequences. When sequencehomology is expressed as a percentage, e.g., 50%, the percentage denotesthe proportion of matches over the length of sequence from hPAK65 thatis compared to some other sequence. Gaps (in either of the twosequences) are permitted to maximize matching; gap lengths of 15 basesor less are usually used, 6 bases or less are preferred with 2 bases orless more preferred. When using oligonucleotides as probes or treatmentsthe sequence homology between the target nucleic acid and theoligonucleotide sequence is generally not less than 17 target basematches out of 20 possible oligonucleotide base pair matches (85%);preferably not less than 9 matches out of 10 possible base pair matches(90%), and most preferably not less than 19 matches out of 20 possiblebase pair matches (95%).

The term "selectively hybridize" referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsof the invention selectively hybridize to nucleic acid strands underhybridization and wash conditions that minimize appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyConditions can be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, andfragments of the invention and a nucleic acid sequence of interest willbe at least 80%, and more typically with preferably increasinghomologies of at least 85%, 90%, 95%, 99%, and 100%.

Two amino acid sequences are homologous if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least 30 amino acids in length) are homologous, as thisterm is used herein, if they have an alignment score of at more than 5(in standard deviation units) using the program ALIGN with the mutationdata matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., inAtlas of Protein Sequence and Structure, 1972, volume 5, NationalBiomedical Research Foundation, pp. 101-110, and Supplement 2 to thisvolume, pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program.

The term "corresponds to" is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term "complementary to" is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence "TATAC" corresponds to a reference sequence "TATAC"and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: "reference sequence", "comparisonwindow", "sequence identity", "percentage of sequence identity", and"substantial identity". A "reference sequence" is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing such as a SEQUENCE IDNO 1, or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a"comparison window" to identify and compare local regions of sequencesimilarity. A "comparison window", as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted by the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48: 443, by the search for similarity method of Pearson andLipman (1988) Proc. Nati. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected. Theterm "sequence identity" means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term "percentage of sequence identity" is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms "substantial identity" as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the,reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thehuman PAK65 polynucleotide sequence shown in FIG. 2A.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage (Immunology--A Synthesis, 2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991), which is incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention. Similarly,unless specified otherwise, the lefthand end of single-strandedpolynucleotide sequences is the 5' end; the lefthand direction ofdouble-stranded polynucleotide sequences is referred to as the 5'direction. The direction of 5' to 3' addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5' to the5' end of the RNA transcript are referred to as "upstream sequences";sequence regions on the DNA strand having the same sequence as the RNAand which are 3' to the 3' end of the RNA transcript are referred to as"downstream sequences".

As applied to polypeptides, the term "substantial identity" means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfurcontaining side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-gltitamine.

The term "polypeptide fragment" as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence (e.g., the cDNA sequence shown in FIG. 2A).Fragments typically are at least 5, 6, 8 or 10 amino acids long,preferably at least 14 amino acids long, more preferably at least 20amino acids long, usually at least 50 amino acids long, and even morepreferably at least 70 amino acids long.

The term "analog" as used herein refers to polypeptides which arecomprised of a segment of at least 25 amino acids that has substantialidentity to a portion of the deduced amino acid sequence shown in FIG.2A and which has at least one of the following properties: (1) specificbinding to a p21 polypeptide, preferably rac1 or CDC42Hs, under suitablebinding conditions, (2) ability to effectuate a p21 protein activity,preferably rac1 or CDC42Hs activity, when expressed in a mammalian cell,(3) serine protein kinase activity, or (4) ability to modulate p21protein activity, preferably rac1 or CDC42Hs GTPase activity. Typically,analog polypeptides comprise a conservative amino acid substitution (oraddition or deletion) with respect to the naturally-occurring sequence.Analogs typically are at least 20 amino acids long, preferably at least50 amino acids long or longer, most usually being as long as full-lengthnaturally-occurring hPAK65 polypeptide as shown in FIG. 2A. Somep21-interacting hPAK65 polypeptide analogs may lack biological activitybut may still be employed for various uses, such as for raisingantibodies to p21-interacting PAK polypeptide epitopes, as animmunological reagent to detect and/or purify p21-interacting PAKpolypeptide antibodies by affinity chromatography, or as a competitiveor noncompetitive agonist, antagonist, or partial agonist of nativep21-interacting hPAK65 polypeptide function.

The term "modulation of human PAK65" is used herein to refer to thecapacity to either enhance or inhibit a functional property of humanPAK65 (e.g., kinase activity, p21-binding); such enhancement orinhibition may be contingent on the occurrence of a specific event, suchas activation of a signal transduction pathway, and/or may be manifestonly is particular cell types.

The term "agent" is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues. Agents are evaluated forpotential activity as human PAK65 modulatory agents (e.g.,antineoplastic agents, cytotoxic agents, inhibitors of neoplastictransformation or cell proliferation, cell proliferation-promotingagents, Ras-induced tumorigenicity, and the like) by inclusion inscreening assays described herein.

The term "candidate agent" is used herein to refer to an agent which isidentified by one or more screening method(s) of the invention as aputative human PAK65 modulatory agent. Some candidate modulatory agentshave therapeutic potential as drugs for human use.

As used herein, the terms "label" or "labeled" refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, ¹³¹ I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

As used herein, "substantially pure" means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term "pharmaceutical agent or drug" as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated hereinby reference).

The term "antineoplastic agent" is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

Detection Of hPAK65 Nucleic Acid Using hPAK65 Polynucleotides

The isolated polynucleotides of the invention can be used as probes todetect, and if desired to clone, PAK65 in a sample from an organism. Thesample is typically from tissue. (Sambrook, J., Fritsch, E. F., andManiatis, T., Molecular Cloning, second edition (1989) (referred toherein as "Sambrook et al.") Isolated polynucleotides with nucleic acidsequences encoding human PAK65 (e.g. SEQ ID NO: 1) can be used as probesto detect the presence of target nucleic acid sequences with sequencehomology. Polynucleotide probes are prepared and labelled by methodsknown in the art, e.g., Sambrook et al, especially chapter 10,the textof sections on preparing and labelling nucleic acids is hereinincorporated by reference. For example, the polymerase chain reactioncan be used to amplify the DNA and a biotin-avidin label system can beused to label and detect the target polynucleotide. Polynucleotideprobes are hybridized with target nucleic acids at appropriatehybridization temperatures (e.g. see Sambrook et al; the text of Chapter9 is herein incorporated by reference); and washed at low and high washstringencies, depending on the detection assay.

Preferably polynucleotides are used as probes under high stringency washconditions and with corresponding hybridization conditions, as known inthe art. Isolated polynucleotides can be used to make probes that are 50base pairs to the full length of hPAK65 cDNA. Preferably probes are madefrom isolated polynucleotides 100-400 nucleotides in length. Suchconditions can be used to detect alleles of the hPAK65 gene in humans.

Alternatively, oligonucleotides can be employed as probes. Techniquesfor using oligonucleotides as probes to detect the same or relatednucleic acid sequences is well known in the art, see for exampleSambrook et al, especially Chapter 11, the text of which is hereinincorporated by reference. Probes can be made from oligonucleotides thatare 10 to 200 bases in length. Preferably probes are made fromoligonucleotides 10 to 60 nucleotides in length and most preferably 12to 40 bases in length. To decrease the number of false positives,preferably two probes are used to identify clones that bind to bothprobes under hybridization and wash conditions. Oligonucleotides can besynthesized on an Applied BioSystems oligonucleotide synthesizeraccording to specifications provided by the manufacturer.

One method for amplification of target nucleic acids, for later analysisby hybridization assays, is known as the polymerase chain reaction("PCR") or PCR technique. The PCR technique can be applied to detectsequences of the invention in suspected samples using oligonucleotideprimers spaced apart from each other and based on the genetic sequenceset forth herein. The primers are complementary to opposite strands of adouble stranded DNA molecule and are typically separated by from about50 to 450 nucleotides or more (usually not more than 2000 nucleotides).This method entails preparing the specific oligonucleotide primersfollowed by repeated cycles of target DNA denaturation, primer binding,and extension with a DNA polymerase to obtain DNA fragments of theexpected length based on the primer spacing. Extension productsgenerated from one primer serve as additional target sequences for theother primer. The degree of amplification of a target sequence iscontrolled by the number of cycles that are performed and istheoretically calculated by the simple formula 2n where n is the numberof cycles. Given that the average efficiency per cycle ranges from about65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of thetarget sequence. The PCR method is described in a number ofpublications, including Saiki et al., Science (1985) 230: 1350-1354;Saiki et al., Nature (1986) 324: 163-166; and Scharf et al., Science(1986) 233: 1076-1078. Also see U.S. Pat. Nos. 4,633,194; 4,683,195; and4,683,202, the text of each patent is herein incorporated by reference.Additional methods for PCR amplification are described in: PCRTechnology: Principles and Applications for DNA Amplification ed. HAErlich, Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide toMethods and Applications, eds. Innis, Gelfland, Snisky, and White,Academic Press, San Diego, Calif. (1990); Mattila et al. (1991) NucleicAcids Res. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methodsand Applications 1: 17, and; PCR, eds. McPherson, Quirkes, and Taylor,IRL Press, Oxford, which are incorporated herein by reference.

Vectors suitable for replication in mammalian cells are known in theart, and can include viral replicons, or sequences that ensureintegration of the sequence encoding PAK65 into the host genome.Suitable vectors can include, for example, those derived from simianvirus SV40, retroviruses, bovine papilloma virus, vaccinia virus, andadenovirus.

A suitable vector, for example, is one derived from vaccinia viruses. Inthis case, the heterologous DNA is inserted into the vaccinia genome.Techniques for the insertion of foreign DNA into the vaccinia virusgenome are known in the art, and utilize, for example, homologousrecombination. The insertion of the heterologous DNA is generally into agene which is non-essential in nature, for example, the thymidine kinasegene (tk), which also provides a selectable marker. Plasmid shuttlevectors that greatly facilitate the construction of recombinant viruseshave been described (see, for example, Mackett et al. (1984);Chakrabarti et al. (1985); Moss (1987)). Expression of the heterologouspolypeptide then occurs in cells or individuals which are immunized withthe live recombinant vaccinia virus.

Such suitable mammalian expression vectors usually contain one or moreeukaryotic transcription units that are capable of facilitatingexpression in mammalian cells. The transcription unit is comprised of atleast a promoter element to mediate transcription of foreign DNAsequences. Suitable promoters for mammalian cells are known in the artand include viral promoters such as that from simian virus 40 (SV40),cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus (ADV), andbovine papilloma virus (BPV).

The optional presence of an enhancer element (enhancer), combined withthe promoter elements described herein, will typically increaseexpression levels. An enhancer is any regulatory DNA sequence that canstimulate transcription up to 1000-fold when linked to endogenous orheterologous promoters, with synthesis beginning at the normal mRNAstart site. Enhancers are also active when they are placed upstream ordownstream from the transcription initiation site, in either normal orflipped orientation, or at a distance of more than 1000 nucleotides fromthe promoter (Maniatis et al. (1987) Science 236: 1237; Alberts et al.(1989) Molecular Biology of the Cell, 2nd ed.). Enhancer elementsderived from viruses can be particularly useful, because they typicallyhave a broader host range. Examples useful in mammalian cells includethe SV40 early gene enhancer (Dijkema et al (1985) EMBO J. 4: 761) andthe enhancer/promoters derived from the long terminal repeat (LTR) ofthe Rous Sarcoma Virus (Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777) and from human cytomegalovirus (Boshart. et al. (1985) Cell 41:521). Additionally, some enhancers are regulatable and become activeonly in the presence of an inducer, such as a hormone or metal ion(Sassone-Corsi and Borelli (1986) Trends Genet. 2: 215; Maniatis et al.(1987) Science 236: 1237).

In addition, the transcription unit can also be comprised of atermination sequence and poly(A) addition sequences which are operablylinked to the PAK65 coding sequence. The transcription unit can also becomprised of an enhancer sequence which increases the expression ofPAK65.

Sequences that cause amplification of the gene may also be desirable, asare sequences which encode selectable markers. Selectable markers formammalian cells are known in the art, and include for example, thymidinekinase, dihydrofolate reductase (together with methotrexate as a DHFRamplifier), aminoglycoside phosphotransferase, hygromycin Bphosphotransferase, asparagine synthetase, adenosine deaminase,metallothionien, and antibiotic resistant genes such as neomycin.

The vector that encodes PAK65 can be used for transformation of asuitable mammalian host cell. Transformation can be by any known methodfor introducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus and transducing a host cell withthe virus or by transfection procedures known in the art, as exemplifiedby U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (thesepatents are incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells areknown in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines. As discussed below, celllines of particular preference are those expressing recombinant hPAK65constructs having constitutive serine kinase activity, and which morepreferably subsequently develop characteristics of a transformed cell.

In the case of expression in insect cells, generally the components ofthe expression system include a transfer vector, usually a bacterialplasmid, which contains both a fragment of the baculovirus genome, and aconvenient restriction site for insertion of the heterologous gene orgenes to be expressed; a wild type baculovirus with a sequencehomologous to the baculovirus-specific fragment in the transfer vector(this allows for the homologous recombination of the heterologous genein to the baculovirus genome); and appropriate insect host cells andgrowth media. A preferred vector is pAcC13 (Rubinfeld, B. (1991) Cell65: 1033-1042). A preferred expression plasmid is pAcPAK780, whichcontains a Myc-epitope:hPAK65 fusion construct. A preferred insect cellline is Sf9. The nucleotide sequence of the coding region of theMyc-epitope:hPAk65 joint region of pAcPAK780 is ATG GAG CAG AAG CTG ATCTCC GAG GAG GAC CTG ATG GAG GAA, which continues to the end of thehPAK65 coding sequence in FIG. 2A.

One of the most commonly used transfer vector for introducing foreigngenes into AcNPV is pAc373. Many other vectors, known to those of skillin the art, have also been designed. These include, for example, pVL985(which alters the polyhedrin start codon from ATG to ATT, and whichintroduces a BamHI cloning site 32 base pairs downstream from the ATT;see Luckow and Summers, Virology (1989) 17: 31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42: 177) and a procaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5' to 3')transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5' end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus transfer vector can alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression can be either regulated orconstitutive.

Additionally, the PAK65 polynucleotide or a fragment thereof can beexpressed in a bacterial system. Therein, a bacterial promoter is anyDNA sequence capable of binding bacterial RNA polymerase and initiatingthe downstream (3") transcription of a coding sequence (e.g. structuralgene) into mRNA. A promoter will have a transcription initiation regionwhich is usually placed proximal to the 5' end of the coding sequence.This transcription initiation region typically includes an RNApolymerase binding site and a transcription initiation site. A bacterialpromoter can also have a second domain called an operator, that canoverlap an adjacent RNA polymerase binding site at which RNA synthesisbegins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein can bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression can occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation can be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5') to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli) (Raibaud et al. (1984) Annu. Rev. Genet. 18: 173]. Regulatedexpression can therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (Chang etal. (1977) Nature 198: 1056), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) (Goeddel et al. (1980) Nuc. Acids Res. 8: 4057; Yelverton et al.(1981) Nucl. Acids Res. 9: 731; U.S. Pat. No. 4,738,921; EPO Pub. Nos.36,776 and 121,775). The β-lactomase (bla) promoter system (Weissmann(1981). In Interferon 3 (ed. I. Gresser)), bacteriophage lambda PL(Shimatake et al. (1981) Nature 292: 128) and T5 (U.S. Pat. No.4,689,406) promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter can be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc.Natl. Acad. Sci. 80: 21). Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce high levels of expression ofsome genes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system (Studier et al. (1986)J. Mol. Biol. 189: 113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EPO Pub. No.267,851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of the PAK65 gene orfragment thereof in prokaryotes. In E. coli, the ribosome binding siteis called the Shine-Dalgarno (SD) sequence and includes an initiationcodon (ATG) and a sequence 3-9 nucleotides in length located 3-11nucleotides upstream of the initiation codon (Shine et al. (1975) Nature254: 34). The SD sequence is thought to promote binding of mRNA to theribosome by the pairing of bases between the SD sequence and the 3' andof E. coli 16S rRNA (Steitz et al. (1979). In Biological Regulation andDevelopment: (Gene Expression (ed. R. F. Goldberger)). To expresseukaryotic genes and prokaryotic genes with weak ribosome-binding site(Sambrook et al. (1989) "Expression of cloned genes in Escherichiacoli." In Molecular Cloning: A Laboratory Manual).

PAK65 can be expressed intracellularly. A promoter sequence can bedirectly linked with the PAK65 gene or a fragment thereof, in which casethe first amino acid at the N-terminus will always be a methionine,which is encoded by the ATG start codon. If desired, methionine at theN-terminus can be cleaved from the protein by in vitro incubation withcyanogen bromide or by either in vivo on in vitro incubation with abacterial methionine N-terminal peptidase (EPO Pub. No. 219,237).

Fusion proteins provide an alternative to direct expression. Typically,a DNA sequence encoding the N-terminal portion of an endogenousbacterial protein, or other stable protein, is fused to the 5' end ofheterologous PAK65 coding sequences. Upon expression, this constructwill provide a fusion of the two amino acid sequences. For example, thebacteriophage lambda cell gene can be linked at the 5' terminus of thePAK65 gene or fragment thereof and expressed in bacteria. The resultingfusion protein preferably retains a site for a processing enzyme (factorXa) to cleave the bacteriophage protein from the PAK65 gene or fragmentthereof (Nagai et al. (1984) Nature 309: 810). Fusion proteins can alsobe made with sequences from the lacZ (Jia et al. (1987) Gene 60: 197),trpE (Allen et al. (1987) J. Biotechnol. 5: 93; Makoff et al. (1989) J.Gen. Microbiol. 135: 11), and Chey (EPO Pub. No. 324,647) genes. The DNAsequence at the junction of the two amino acid sequences may or may notencode a cleavable site. Another example is a ubiquitin fusion protein.Such a fusion protein is made with the ubiquitin region that preferablyretains a site for a processing enzyme (e.g. ubiquitin specificprocessing-protease) to cleave the ubiquitin from the PAK65 polypeptide.Through this method, mature PAK65 polypeptides can be isolated (Milleret al. (1989) Bio/Technology 7: 698). A preferred recombinantly derivedfusion contains an Myc-epitope (with an added methionine: MEQKLISEEDL)fused to the N-terminal of a PAK polypeptide or fragment. An antibodyspecific for this Myc epitope allows isolation and or identification (asin an assay) of the fusion protein. One such embodiment is the constructfound inhPAK65 expression vector pAcPAK780. Another preferred system isa fusion with glutathione-S-transferase ("GST"; available fromPharmacia) at the C-terminal end of hPAK65 or its fragment, Therecombinant fusion protein is readily isolated by its ability to bind toglutathione attached to solid support followed by elution of the fusionwith glutathione. A most preferred embodiment of this type is found inplasmid pGSTPAKette which encodes the recombinant fusion proteinreferred to as GST-PAKette, which contains GST fused to a hPAK65 proteinfragment amino acids 49 to 113 and which retains the ability tospecifically bind rac1 or CDC42Hs. This domain of hPAK65 contains theraci binding domain. The amino acid sequence of GST-PAKette is providedin FIG. 10.

Alternatively, hPAK65 polypeptides can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of thehPAK65 polypeptides in bacteria (U.S. Pat. No. 4,336,336). The signalsequence fragment typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and thePAK65 polypeptide.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) (Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3: 2437) and the E. colialkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc.Natl. Acad. Sci. 82: 7212). As an additional example, the signalsequence of the alpha-amylase gene from various Bacillus strains can beused to secrete heterologous proteins from B. subtilis (Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79: 5582; EPO Pub. No. 244,042).

Typically, transcription termination sequences recognized by bacteriaare regulatory regions located 3' to the translation stop codon, andthus together with the promoter flank the coding sequence. Thesesequences direct the transcription of an mRNA which can be translatedinto the polypeptide encoded by the DNA. Transcription terminationsequences frequently include DNA sequences of about 50 nucleotidescapable of forming stem loop structures that aid in terminatingtranscription. Examples include transcription termination sequencesderived from genes with strong promoters, such as the trp gene in E.coli as well as other biosynthetic genes.

Typically, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (e.g., plasmids) capable of stable maintenancein a host, such as bacteria. The replicon will have a replicationsystem, thus allowing it to be maintained in a procaryotic host eitherfor expression or for cloning and amplification. In addition, a repliconcan be either a high or low copy number plasmid. A high copy numberplasmid will generally have a copy number ranging from about 5 to about200, and typically about 10 to about 150. A host containing a high copynumber plasmid will preferably contain at least about 10, and morepreferably at least about 20 plasmids. Either a high or low copy numbervector can be selected, depending upon the effect of the vector andthehPAK65 polypeptide on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. For example, integrating vectors constructed withDNA from various Bacillus strains integrate into the Bacillus chromosome(EPO Pub. No. 127,328). Integrating vectors can also be comprised ofbacteriophage or transposon sequences.

Typically, extrachromosomal and integrating expression constructs cancontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and can include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev.Microbiol. 32: 469). Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis (Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79: 5582; EPO Pub. Nos. 36,259 and63,953; PCT WO 84/04541), Escherichia coli (Shimatake et al. (1981)Nature 292: 128; Amann et al. (1985) Gene 40: 183; Studier et al. (1986)J. Mol. Biol. 189: 113; EPO Pub. Nos. 36,776, 136,829 and 136,907; UKPatent Application Serial No. 8418273), Streptococcus cremoris (Powellet al. (1988) Appl. Environ. Microbiol. 54: 655) Streptococcus lividans(Powell et al. (1988) Appl. Environ. Microbiol. 54: 655), Streptomyceslividans (U.S. Pat. No. 4,745,056).

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and typically include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See e.g., (Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79: 5582; EPO Pub.Nos. 36,259 and 63,953; P.C.T. WO 84/04541, Bacillus), (Miller et al.(1988) Proc. Natl. Acad. Sci. 85: 856; Wang et al. (1990) J. Bacteriol.172: 949, Campylobacter), (Cohen et al. (1973) Proc. Natl. Acad. Sci.69: 2110; Dower et al. (1988) Nucleic Acids Res. 16: 6127; Kushner(1978) "An improved method for transformation of Escherichia coli withColE1-derived plasmids. In Genetic Engineering: Proceedings of theInternational Symposium on Genetic Engineering (eds. H. W. Boyer and S.Nicosia); Mandel et al. (1970) J. Mol. Biol. 53: 159; Taketo (1988)Biochim. Biophys. Acta 949: 318; Escherichia), (Chassy et al. (1987)FEMS Microbiol. Lett. 44: 173 Lactobacillus); (Fiedler et al. (1988)Anal. Biochem 170: 38, Pseudomonas); (Augustin et al. (1990) FEMSMicrobiol. Lett. 66: 203, Staphylococcus), (Barany et al. (1980) J.Bacteriol. 144: 698; Harlander (1987) "Transformation of Streptococcuslactis by electroporation," in: Streptococcal Genetics (ed. J. Ferrettiand R. Curtiss III); Perry et al. (1981) Infec. Immun. 32: 1295; Powellet al. (1988) Appl. Environ. Microbiol. 54: 655; Somkuti et al. (1987)Proc. 4th Evr. Cong. Biotechnology 1: 412, Streptococcus).

As discussed herein, minor variations in the amino acid sequence ofhPAK65 protein are contemplated as being encompassed by the term hPAK65,providing that the minor variations in the amino acid sequence maintainat least 95%, more preferably at least 99%, and to at least one aminoacid difference in homology to the human PAK65 protein encoding sequencepresented in FIG. 2A. Such minor variations are likely to occur amongstallelic variants of hPAK65. In particular, conservative amino acidreplacements are contemplated. Conservative replacements are those thattake place within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are generally divided intofamilies: (1) acidic=aspaitate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Morepreferred families are: serine and threonine are aliphatic-hydroxyfamily; asparagine and glutamine are an amide-containing family;alanine, valine, leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family. Forexample, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding or kinase properties of the resulting molecule, especially ifthe replacement does not involve an amino acid at a p21-binding site orkinase active site. Whether an amino acid change results in a functionalpeptide can readily be determine assaying the specific activity of thepolypeptide derivative. Assays are described in detail herein.

Fragments or analogs of hPAK65 can be prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. For example, suchfunctional domains include domains conferring the property of binding toform an hPAK65-p21 protein complex, protein kinase domain,autophosphorylation domain, and domains conferring the property ofmodulating hPAK65-related signal transduction pathways of cells.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function, such asthe kinase domain as provided in the Example section. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known (Bowie et al. (1991) Science 253: 164). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in an hPAK65 sequence.

Fragments or analogs comprising substantially one or more functionaldomains may be fused to heterologous potypeptide sequences, wherein theresultant fusion protein exhibits the functional properties conferred bythe hPAK65 fragment. For example, fusion protein GST-PAKette (containingamino acids 49 to 113) retains the ability to specifically bind rac1 andCDC42Hs. Alternatively, polypeptides wherein one or more functionaldomain have been deleted will exhibit a loss of the property normallyconferred by the missing fragment. For example, an hPAK65 N-terminaltruncation protein containing nucleotides 1065-2248 did not retainserine kinase activity.

Although one class of preferred embodiments are fragments having amino-and/or carboxy-termini corresponding to amino acid positions nearfunctional domains borders, alternative fragments may be prepared. Thechoice of the amino- and carboxy-termini of such fragments rests withthe discretion of the practitioner and will be made based onexperimental considerations such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, or other considerations.

In addition to fragments, analogs of hPAK65 can be made. Such analogsmay include one or more deletions or additions of amino acid sequence,either at the amino- or carboxy-termini, or internally, or both; analogsmay further include sequence transpositions. Analogs may also compriseamino acid substitutions, preferably conservative substitutions.Additionally, analogs may include heterologous sequences generallylinked at the amino-, or carboxy-terminus, wherein the heterologoussequence(s) confer a functional property to the resultant analog whichis not inherent to a native hPAK65 protein. However, analogs mustcomprise a segment of 25 amino acids that has substantial identity to aportion of the native protein amino acid sequence. Preferred amino acidsubstitutions are those which: (1) reduce susceptibility to proteolysis,(2) reduce susceptibility to oxidation, (3) alter binding-affinity forforming hPAK65-p21 protein complexes, (4) alter binding affinity forforming hPAK65 complexes to proteins subtrates for serine kinaseactivity, and (4) confer or modify other physicochemical or functionalproperties of such analogs. Analogs can include various muteins of ahPAK65 sequence other than the naturally-occurring peptide sequence. Forexample, single or multiple amino acid substitutions (preferablyconservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts.

A conservative amino acid substitution should not substantially changethe structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W. H.Freeman and Company, New York; Introduction to Protein Structure,(1991), C. Branden and J. Tooze, Garland Publishing, New York, N.Y.; andThornton et al. (1991) Nature 354: 105; which are incorporated herein byreference).

It can be advantageous to employ a peptide analog of hPAK65, or aportion thereof, as a pharmaceutical agent or as a commercial researchreagent. For example, a peptide analog of hPAK65 having high affinityfor binding a p21 protein can be used as a competitive inhibitor ofhPAK65-p21 protein complex formation by competing with native hPAK65 lorbinding to a p21 protein.

In addition to polypeptides consisting only of naturally-occurring aminoacids, peptidomimetics are also provided. Peptide analogs are commonlyused in the pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompound are termed "peptide mimetics" or "peptidomimetics" (Fauchere,J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392;and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporatedherein by reference) and are usually developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce anequivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human PAK65, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:--CH₂ NH--, --CH₂ S--, --CH₂ --CH₂ --, --CH═CH-- (cis and trans),--COCH₂ --, --CH(OH)CH₂ --, and --CH₂ SO--, by methods known in the artand further described in the following references: Spatola, A. F. in"Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B.Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F.,Vega Data (March 1983), Vol. 1, Issue. 3, "Peptide BackboneModifications" (general review); Morley, J. S., Trends Pharm Sci (1980)pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res(1979) 14: 177-185 (--CH₂ NH--, CH₂ CH₂ --); Spatola, A. F. et al., LifeSci (1986) 38: 1243-1249 (--CH₂ --S); Hann, M. M., J Chem Soc PerkinTrans I (1982) 307-314 (--CH--CH--, cis and trans); Almquist, R. G. etal., J Med Chem (1980) 23: 1392-1398 (--COCH₂ --); Jennings-White, C. etal., Tetrahedron Lett (1982) 23: 2533 (--COCH₂ --); Szelke, M. et al.,European Appln. EP 45665 (1982) CA: 97: 39405 (1982) (--CH(OH)CH₂ --);Holladay, M. W. et al., Tetrahedron Lett (1983) 24: 4401-4404(--C(OH)CH₂ --); and Hruby, V. J., Life Sci (1982) 31: 189-199 (--CH₂--S--); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is --CH₂ NH--. Such peptidemimetics may have significant advantages over polypeptide embodiments,including, for example: more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., are not contact pointsin hPAK65-p21 complexes) to which the peptidomimetic binds to producethe therapeutic effect. Derivitization (e.g., labelling) ofpeptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387,incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

Human PAK65 cDNA sequences are identified and genomic clones can beisolated by screening a human genomic clone library, such as a humangenomic library in yeast artificial chromosomes, cosmids, orbacteriophage λ (e.g., λ Charon 35), with a polynucleotide probecomprising a sequence of about at least 30 contiguous nucleotides (ortheir complement) of the cDNA sequence shown in FIG. 2A. Typically,hybridization and washing conditions are performed at high stringencyaccording to conventional hybridization procedures. Positive clones areisolated and sequenced. For illustration and not for limitation, afull-length polynucleotide corresponding to the sequence of FIG. 2A maybe labeled and used as a hybridization probe to isolate genomic clonesfrom a human genomic clone library in λEMBL4 or λGEM11 (PromegaCorporation, Madison, Wis.); typical hybridization conditions forscreening plaque lifts (Benton and Davis (1978) Science 196: 180) canbe: 50% formamide, 5×SSC or SSPE, 1-5×Denhardt's solution, 0.1-1% SDS,100-200 μg sheared heterologous DNA or tRNA, 0-10% dextran sulfate,1×10⁵ to 1×10⁷ cpm/ml of denatured probe with a specific activity ofabout 1×10⁸ cpm/μg, and incubation at 42° C. for about 6-36 hours.Prehybridization conditions are essentially identical except that probeis not included and incubation time is typically reduced. Washingconditions are typically 1-3×SSC, 0.1-1% SDS, 50-70° C. with change ofwash solution at about 5-30 minutes. Cognate human sequnces, includingallelic sequences, can be obtained in this manner.

Nonhuman cDNAs and genomic clones (i.e., cognate nonhuman PAK65 genes)can be analogously isolated from various nonhuman cDNA and genomic clonelibraries available in the art (e.g., Clontech, Palo Alto, Calif.) byusing probes based on the sequences shown in FIG. 2A, with hybridizationand washing conditions typically being less stringent than for isolationof human clones. A most preferred embodiment is a PAK65 gene from mouse.

Polynucleotides comprising sequences of approximately 30-50 nucleotides,preferably at least 100 nucleotides, corresponding to or complementaryto the nucleotide sequence shown in FIG. 2A can serve as PCR primers (atleast 10 nucleotides) and/or hybridization probes for identifying andisolating germline genes closely related to hPAK65. These germline genesmay be human or may be from another mammalian species, preferablyprimates or mice. Such germline genes may be isolated by various methodsconventional in the art, including, but not limited to, by hybridizationscreening of genomic libraries in bacteriophage λ or cosmid libraries,or by PCR amplification of genomic sequences using primers derived fromthe sequences shown in FIG. 2A. Human genomic libraries are publiclyavailable or may be constructed de novo from human DNA.

Genomic clones of PAK65, particularly of the murine cognate PAK65 gene,may be used to construct homologous targeting constructs for generatingcells and transgenic nonhuman animals having at least one functionallydisrupted PAK65 allele, preferably homozygous for ablated PAK65 alleles.Guidance for construction of homologous targeting constructs may befound in the art, including: Rahemtulla et al. (1991) Nature 353: 180;Jasin et al. (1990) Genes Devel. 4: 157; Koh et al. (1992) Science 256:1210; Molina et al. (1992) Nature 357: 161; Grusby et al. (1991) Science253: 1417; Bradley et al. (1992) Bio/Technology 10: 534, incorporatedherein by reference). Homologous targeting can be used to generateso-called "knockout" mice, which are heterozygous or homozygous for aninactivated PAK65 allele. Such mice may be sold commercially as researchanimals for investigation of PAK65-dependent conditions, such as cellstructural integrity, neoplasia, cell proliferation, signaltransduction, drug screening, and other uses.

Chimeric targeted mice are derived according to Hogan, et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C.,(1987) which are incorporated herein by reference. Embryonic stem cellsare manipulated according to published procedures (Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRLPress, Washington, D.C. (1987); Zjilstra et al. (1989) Nature 342: 435;and Schwartzberg et al. (1989) Science 246: 799, each of which isincorporated herein by reference).

Additionally, a PAK65 cDNA or genomic gene copy may be used to constructtransgenes for expressing PAK65 polypeptides at high levels and/or underthe transcriptional control of transcription control sequences which donot naturally occur adjacent to the PAK65 gene, as discussed above. Forexample but not limitation, a constitutive promoter (e.g., a HSV-tk orpgk promoter) or a cell-lineage specific transcriptional regulatorysequence (e.g., an albumin, elastase, or CD4 or CD8 genepromoter/enhancer) may be operably linked to a PAK65-encodingpolynucleotide sequence to form a transgene (typically in combinationwith a selectable marker such as a neo gene expression cassette). Suchtransgenes can be introduced into cells (e.g., ES cells, hematopoieticstem cells, cultured primary hepatocytes) and transgenic cells andtransgenic nonhuman animals may be obtained according to conventionalmethods. Transgenic cells and/or transgenic nonhuman animals may be usedto screen for antineoplastic agents and/or to screen for potential cellproliferation modulating agents, as overexpression of PAK65 orinappropriate expression of PAK65 may result in a hyperproliferativestate or hypoproliferative state.

The antisense oligonucleotides of the invention can be synthesized byany of the known chemical oligonucleotide synthesis methods. Suchmethods are generally described, for example, in Winnacker, From Genesto Clones: Introduction to Gene Technology. VCH Verlagsgesellschaft mbH(H., Ibelgaufts trans. 1987). Any of the known methods ofoligonucleotide synthesis can be utilized in preparing the instantantisense oligonucleotides. The antisense oligonucleotides are mostadvantageously prepared by utilizing any of the commercially available,automated nucleic acid synthesizers. The device utilized to prepare theoligonucleotides described herein, the Applied Biosystems 380B DNASynthesizer, utilizes β-cyanoethyl phosphoramidite chemistry. Antisenseoligonucleotides hybridizable with any portion of the mRNA transcriptcan be prepared by the oligonucleotide synthesis methods known to thoseskilled in the art. While any length oligonucleotide can be utilized inthe practice of the invention, sequences shorter than 12 bases may beless specific in hybridizing to the target PAK65 mRNA, and may be moreeasily destroyed by enzymatic digestion. Hence, oligonucleotides having12 or more nucleotides are preferred. Sequences longer than 18 to 21nucleotides may be somewhat less effective in inhibiting PAK65translation because of decreased uptake by the target cell. Thus,oligomers of 12-21 nucleotides are most preferred in the practice of thepresent invention, particularly oligomers of 12-18 nucleotides.Oligonucleotides complementary to and hybridizable with any portion ofthe PAK65 mRNA transcript are, in principle, effective for inhibitingtranslation of the transcript, and capable of inducing the effectsherein described. Translation is most effectively inhibited by blockingthe mRNA at a site at or near the initiation codon. Thus,oligonucleotides complementary to the 5'-terminal region of the PAK65mRNA transcript are preferred. Secondary or tertiary structure whichmight interfere with hybridization is minimal in this region. Moreover,sequences that are too distant in the 3' direction from the initiationsite can be less effective in hybridizing the mRNA transcripts becauseof a "read-through" phenomenon whereby the ribosome is postulated tounravel the antisense/sense duplex to permit translation of the message.(see, e.g. Shakin, J. Biochemistry 261, 16018 (1986)). The antisenseoligonucleotide is preferably directed to a site at or near the ATGinitiation codon for protein synthesis. Oligonucleotides complementaryto a portion of the PAK65 mRNA including the initiation codon arepreferred. While antisense oligomers complementary to the 5'-terminalregion of the PAK65 transcript are preferred, particularly the regionincluding the initiation codon, it should be appreciated that usefulantisense oligomers are not limited to those complementary to thesequences found in the translated portion of the mRNA transcript, butalso includes oligomers complementary to nucleotide sequences containedin, or extending into, the 5'- and 3'-untranslated regions. Antisensenucleotides or antisense expression constructs can find use to screenfor antineoplastic agents and/or to screen for potential cellproliferation modulating agents, as inappropriate expression of PAK65may result in a hyperproliferative state or hypoproliferative state.

As been determined herein, there exists a human protein, referred to ashPAK65, which is a 65 kDa protein (as determined by SDS-PAGE underreducing conditions; molecular weight based on deduced amino acidsequence is 56.5 kDa) which interacts in a GTP dependent manner withrac1 and CDC42Hs but not with rho A. The human PAK65 mRNA isubiquitously expressed in human tissues. Recombinant hPAK65 exhibitsidentical specificity as the endogenous p65; both can bind rac1 andCDC42Hs in a GTP dependent manner. The GTP bound forms of rac1 andCDC42Hs induce autophosphorylation of hPAK65 on serine residues only.hPAK65 activated either by rac1 or CDC42HS is phosphorylated on the samesites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hsstimulates hPAK65 kinase activity towards proteins, e.g. myelin basicprotein ("MBP"), and once hPAK65 is activated, rac1 or CDC42Hs are nolonger required to keep it active. The affinities of rac1 and CDC42Hsfor the non phosphorylated or phosphorylated hPAK65 were similar. HumanPAK65 had a marginal effect on the intrinsic GTPase activity of CDC42Hsand a more significant inhibition of the GAP p190 stimulated GTPaseactivity. These data are consistent with a model in which hPAK65function as an effector molecule for rac 1 and CDC42Hs.

Accordingly, the hPAK65 nucleic acids and polypeptides of the inventionfind particular use by providing a new protein kinase activity, ineffecting or modulating human rac1 and CDC42Hs related pathways, inidentifying hPAK65-related pathways and disease conditions, and inidentifying agents that effect or modulate hPAK65-activity and relatedpathways. Such p21 protein modulating agents can provide novelchemotherapeutic agents for treatment of neoplasia, lymphoproliferativeconditions, arthritis, angiogenesis, inflammation, autoimmune diseases,apoptosis, and the like.

The present inventors have determined that certain p21 proteins bind tohPAK65 to form a high affinity intermolecular complex underphysiological conditions, and that these complexes are detected bynumerous assays including a modified "overlay assay" and ELISA. p21protein-hPAK65 complexes are targets for agents capable of modulatingcertain p21 protein dependent (preferably rac1 and CDC42Hs) andhPAK65-dependent pathways, and particularly are target for novelchemotherapeutic or chemopreventative antineoplastic andimmunomodulatory agents. For example, agents which alter rac1:hPAK65interactions in neoplastic and/or preneoplastic cells may be developedas potential human therapeutic drugs. Candidate antineoplastic agentsmay be identified by their ability to inhibit rac1:hPAK65 complexformation in vitro and/or in vivo and/or inhibit hPAK65 protein kinasefunction in vitro and/or in vivo (e.g., block the ability of hPAK65 tophosphorylate cellular targets downstream in a hPAK65-dependentpathway). Accordingly, methods of identifying antineoplastic andimmunomodulatory agents are now provided by the invention.

Candidate antineoplastic agents are then tested further forantineoplastic activity in assays which are routinely used to predictsuitability for use as human antineoplastic drugs. Examples of theseassays include, but are not limited to: (1) ability of the candidateagent to inhibit the ability of anchorage-independent transformed cellsto grow in soft agar, (2) ability to reduce tumorigenicity oftransformed cells transplanted into nu/nu mice, (3) ability to reversemorphological transformation of transformed cells, (4) ability to reducegrowth of transplanted tumors in nu/nu mice, (5) ability to inhibitformation of tumors or preneoplastic cells in animal models ofspontaneous or chemically-induced carcinogenesis, and (6) ability toinduce a more differentiated phenotype in transformed cells to which theagent is applied.

Since hPAK65 is abundant in neutrophils, agents which enhance or inhibithPAK65 activity may serve as immunomodulatory agents, for example, toattenuate an inflammatory reaction, graft-versus-host reaction, orautoimmune condition, neurodegenerative diseases, neoplasia, and thelike.

One category of assay in which hPAK65-modulating agents (e.g., candidateantineoplastic agents) may be identified is a binding inhibition assay,wherein agents are individually (or in pools) evaluated for theirability to inhibit formation of a binding complex comprising a p21protein polypeptide) preferably rac 1 or CDC42Hs) bound to a hPAK65polypeptide under aqueous binding conditions in which p21-protein:hPAK65binding occurs in the absence of the agent (see Examples). hPAK65modulating agents (e.g., candidate antineoplastic agent;) can beidentified by screening for agents which interfere with the formation ofor activity of functional p21-protein:hPAK65 complexes. Agents whichinhibit binding of rac1 polypeptides to hPAK65 polypeptides areidentified as hPAK65-modulating agents (e.g., candidate antineoplasticagents).

The screening assays of the present invention may utilize isolated orpurified forms of the assay components (hPAK65 polypeptides and p21protein polypeptides, preferably rac1 and CDC42Hs). This refers topolypeptides of the present invention which have been separated fromtheir native environment (e.g., a cytoplasmic or nuclear fraction of acell) or by recombinant production, to at least about 10-50% purity. Asubstantially pure composition includes such polypeptide(s) or complexesthat are approaching homogeneity, i.e., about 80-90% pure, preferably95-99% pure, and most preferably greater than 99% pure. Preferredembodiments include binding assays which use rac1 or CDC42Hs incombination with hPAK65 polypeptides which are produced by recombinantmethods or chemically synthesized.

Additional preferred embodiments comprise p21 protein or hPAK65 analogsthat have superior stabilities as experimental reagents. For example,preferred analogs may be resistant to degradation by proteolyticactivities present in the binding reaction(s), and/or may be resistantto oxidative inactivation. Such analogs may include amino acidsubstitutions which remove proteolytic cleavage sites and/or replaceresidues responsible for oxidative inactivation (e.g., methionine,cysteine). However, the analogs must be functional in at least thecontrol binding assay(s); therefore, analogs comprising amino acidsubstitutions which destroy or significantly degrade the functionalutility of the analog in the binding assay are not employed for suchassays. A preferred hPAK65 p21 protein binding polypeptide is containshPAK65 amino acids 49 to 113, with a more preferred form as fusionprotein GST-PAKette. A preferred polypeptide for kinase inhibitionassays is a constitutively active hPAK65. Preferred polypeptides of thisare truncations missing the regulatory domain that inhibits kinaseactivity or are glutamic or aspartic acid substitutions for serine 380,serine 383 and/or threonine 384. Preferred p21 protein analogs have anidentifying tag moiety attached, preferably a peptide epitope tagrecognizable by an antibody. Preferred tags are the Myc-epitope andGlu-Glu peptide tags as provided in the Examples. Also preferred arehPAK65 having C-terminal mutations where the amino acids 489-491 (i.e.,Ser-Ser-Leu) are replaced non-conservatively or are removed. Oneconstitutively active hPAK65 embodiment of this type has a deletion ofamino acids 482 to 506. A preferred embodiment has both the previouslydiscussed myc-epitope taged rhPAK65 and the 482-506 deletion.

These methods of screening may involve labelling a p21 protein or hPAK65polypeptide with any of a myriad of suitable markers, includingradiolabels (e.g., ¹²⁵ I or ³² P), various fluorescent labels andenzymes, (e.g., glutathione-S-transferase, luciferase, andβ-galactosidase). If desired for basic binding assays, the targetpolypeptide may be immobilized by standard techniques. For example butnot for limitation, such immobilization may be effected by linkage to asolid support, such as a chromatographic matrix, microtiter plate well,or by binding to a charged surface, such as a Nylon 66 membrane.

Binding assays generally take one of two forms: immobilized hPAK65polypeptide(s) can be used to bind labeled p21 protein polypeptide(s),or conversely, immobilized p21 protein polypeptide(s) can be used tobind labeled hPAK65 polypeptides. In each case, the labeled polypeptideis contacted with the immobilized polypeptide under aqueous conditionsthat permit specific binding of the polypeptides(s) to form a complex inthe absence of added agent. Particular aqueous conditions may beselected by the practitioner according to conventional methods. Forgeneral guidance, the following buffered aqueous conditions may be used:10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition ofdivalent cation(s) and/or metal chelators and/or nonionic detergentsand/or membrane fractions. It is appreciated by those in the art thatadditions, deletions, modifications (such as pH) and substitutions (suchas KCl substituting for NaCl or buffer substitution) may be made tothese basic conditions. Modifications can be made to the basic bindingreaction conditions so long as specific binding of p21 proteinpolypeptide(s) to hPAK65 polypeptides occurs in the control reaction(s).Conditions that do not permit specific binding in control reactions (noagent included) are not suitable for use in inding assays. As determinedherein human rho A does not bind to nor activate hPAK65.

Preferably, at least one polypeptide species is labeled with adetectable marker. Suitable labeling includes, but is not limited to,radiolabeling by incorporation of a radiolabeled amino acid (e.g., ¹⁴C-labeled leucine, ³ H-labeled glycine, ³⁵ S-labeled methionine),radiolabeling by post-translational radioiodination with ¹²⁵ I or ¹³¹ I(e.g., Bolton-Hunter reaction and chloramine T), labeling bypost-translational phosphorylation with ³² P (e.g., phosphorylase andinorganic radiolabeled phosphate), fluorescent labeling by incorporationof a fluorescent label (e.g., fluorescein or rhodamine), or labeling byother conventional methods known in the art. In embodiments where one ofthe polypeptide species is immobilized by linkage to a substrate, theother polypeptide is generally labeled with a detectable marker. Apreferred format is an ELISA asssy, in which a preferred label is apeptide epitope that is specifically recognized by an antibody and thatis attached to one of the proteins, for example by chemical or enzymaticconjugation, or preferably by recombinant DNA methods that yield afusion protein. Radiolabled GTPγS that binds to but is not hydrolyzed byp21 protein GTPases can also be used to label a p21 protein polypeptidein a p21protein-hPAK65 binding assay.

Additionally, in some embodiments a p21 protein or hPAK65 polypeptidemay be used in combination with an accessory protein (e.g., a proteinwhich forms a complex with the polypeptide, preferably in vivo). It ispreferred that different labels are used for each polypeptide species,so that binding of individual and/or heterodimeric and/or multimericcomplexes can be distinguished. For example, a CDC42Hs polypeptide maybe labeled with fluorescein and an accessory polypeptide, e.g p190, maybe labeled with a fluorescent marker that fluorescent with either adifferent excitation wavelength or emission wavelength, or both.Alternatively, double-label scintillation counting may be used, whereina polypeptide is labeled with one isotope (e.g., ³ H) and a secondpolypeptide species is labeled with a different isotope (e.g., ¹⁴ C)that can be distinguished by scintillation counting using discriminationtechniques.

Labeled polypeptide(s) are contacted with immobilized polypeptide(s)under aqueous conditions as described herein. The time and temperatureof incubation of a binding reaction may be varied, so long as theselected conditions permit specific binding to occur in a controlreaction where no agent is present. Preferable embodiments employ areaction temperature of about at least 15° C., more preferably 35 to 42°C., and a time of incubation of approximately at least 15 seconds,although longer incubation periods are preferable so that, in someembodiments, a binding equilibrium is attained. Binding kinetics and thethermodynamic stability of bound p21 protein:hPAK65 complexes,preferably rac1:hPAK65 and CDC42Hs:hPAK65 or where hPAK65 is replaced byGST-PAKette, determine the latitude available for varying the time,temperature, salt, pH, and other reaction conditions. However, for anyparticular embodiment, desired binding reaction conditions can becalibrated readily by the practitioner using conventional methods in theart, which may include binding analysis using Scatchard analysis, Hillanalysis, and other methods (Proteins, Structures and MolecularPrinciples, (1984) Creighton (ed.), W.H. Freeman and Company, New York).

Specific binding of labeled (e.g. peptide tagged) p21 protein or hPAK65polypeptide to immobilized hPAK65 or p21 protein polypeptide,respectively, is determined by including unlabeled competitor protein(s)(e.g., albumin). After a binding reaction is completed, labeledpolypeptide(s) that is/are specifically bound to immobilized polypeptideis detected. For example, after a suitable incubation period forbinding, the aqueous phase containing non-immobilized protein is removedand the substrate containing the immobilized polypeptide species and anylabeled protein bound to it is washed with a suitable buffer, optionallycontaining unlabeled blocking agent(s), and the wash buffer(s) removed.After washing, the amount of detectable label remaining specificallybound to the immobilized polypeptide is determined (e.g., by optical,enzymatic, immunological, autoradiographic or other radiochemicalmethods, or combinations thereof). In a preferred format, the label isan antibody detectable epitope (tag), wherein the anti-tag antibody canthen be detected by methods known in the art, eg. via measuring theenzymatic activity of an alkaline phosphatase conjugated to theantibody.

In some embodiments, addition of unlabeled blocking agents that inhibitnon-specific binding are included. Examples of such blocking agentsinclude, but are not limited to, the following: calf thymus DNA, salmonsperm DNA, yeast RNA, mixed sequence (random or pseudorandom sequence)oligonucleotides of various lengths, bovine serum albumin, nonionicdetergents (NP-40, Tween, Triton X-100, etc.), nonfat dry milk proteins,Denhardt's reagent, polyvinylpyrrolidone, Ficoll, and other blockingagents. Practitioners may, in their discretion, select blocking agentsat suitable concentrations to be included in binding assays; however,reaction conditions are selected so as to permit specific bindingbetween a hPAK65 polypeptide and a p21 protein polypeptide in a controlbinding reaction. Blocking agents are induced to inhibit nonspecificbinding of labeled protein to immobilized protein and/or to inhibitnonspecific binding of labeled polypeptide to the immobilizationsubstrate.

In embodiments where a polypeptide is immobilized, covalent ornoncovalent linkage to a substrate may be used. Covalent linkagechemistries include, but are not limited to, well-characterized methodsknown in the art (Kadonaga and Tijan (1986) Proc. Natl. Acad. Sci.(U.S.A.) 83: 5889, which is incorporated herein by reference). Oneexample, not for limitation, is covalent linkage to a substratederivatized with cyanogen bromide (such as CNBr-derivatized Sepharose4B). It may be desirable to use a spacer to reduce potential sterichindrance from the substrate. Noncovalent bonding of proteins to asubstrate include, but are not limited to, bonding of the protein to acharged surface and binding with specific antibodies.

In one class of embodiments, parallel binding reactions are conducted,wherein one set of reactions serves as control and at least one otherset of reactions include various quantities of agents, mixtures ofagents, or biological extracts, that are being tested for the capacityto inhibit binding of a p21 protein polypeptide to an hPAK65polypeptide. Agents that inhibit binding relative to the controlreaction(s) are thereby identified as hPAK65-modulating agents and/orcandidate antineoplastic agents and/or candidate immunomodulatoryagents.

In one variation, GTPase activity and/or GDP- or GTP-binding activity ofp21 protein, preferably rac 1 or CDC42Hs, is measured as the assayendpoint. The ability of hPAK65 polypeptide to modulate one or more ofthese guanine nucleotide activities of p21 protein serves as the basisfor the screening assay (see Example 1--modified overlay assay; seeExample 12). Test compounds which modulate the activity of hPAK65 tomodulate a p21 protein guanine nucleotide activities are therebyidentified as hPAK65 protein modulators and p21 protein modulators.

Yeast comprising (1) an expression cassette encoding a GAL4 DNA bindingdomain (or GAL4 activator domain) fused to a binding fragment of hPAK65capable of binding to a rac1 polypeptide, (2) an expression cassetteencoding a GAL4 DNA activator domain (or GAL4 binding domain,respectively) fused to a binding fragment of rac1 (or CDC42Hs) capableof binding to a hPAK65 polypeptide, and (3) a reporter gene (e.g.,β-galactosidase) comprising a cis-linked GAL4 transcriptional responseelement can be used for agent screening in a two-hybrid screening assay.Such yeast are incubated with a test agent and expression of thereporter gene (e.g., β-galactosidase) is determined; the capacity of theagent to inhibit expression of the reporter gene as compared to acontrol culture identifies the agent as a candidate hPAK65-modulatingagent or p21 protein-modulating agent.

hPAK65 and rac1 or CDC42Hs polypeptides, especially those portions whichform direct contacts in complex, can be used for rational drug design ofcandidate modulating agents (e.g., antineoplastics andimmunomodulators). The substantially purified complexes and theidentification of rac1 or CDDC42Hs as a binding partner for hPAK65 asprovided herein permits production of substantially pure polypeptidecomplexes and computational models which can be used for protein X-raycrystallography or other structure analysis methods, such as the DOCKprogram (Kuntz et al. (1982) J. Mol. Biol. 161: 269; Kuntz ID (1992)Science 257: 1078) and variants thereof. Potential therapeutic drugs maybe designed rationally on the basis of structural information thusprovided. In one embodiment, such drugs are designed to preventformation of a protein complex.

Thus, the present invention may be used to design drugs, including drugswith a capacity to inhibit binding of p21 proteins, preferably rac1 orCDC42Hs, to hPAK65. Using the methods as taught herein other p21proteins are readily tested for there ability to bind to and activatehPAK65.

The design of compounds that interact preferentially with a hPAK65polypeptide or hPAK65: p21 protein complex can be developed usingcomputer analysis of three-dimensional structures. A set of molecularcoordinates can be determined using: (1) crystallographic data, (2) dataobtained by other physical methods, (3) data generated by computerizedstructure prediction programs operating on the deduced amino acidsequence data, or, preferably, a combination of these data. Examples ofphysical methods that may be used to define structure are, for example,two-dimensional homonuclear correlated spectroscopy (COSY). For thoseskilled in the art with one-dimensional NMR spectroscopy, COSY providesthe kind of information available from a single-frequency decouplingexperiment (e.g., which spins are scalar coupled to one another). In aCOSY plot, the ID spectrum lies along the diagonal, and the off-diagonalelements are present at the intersection of chemical shifts of groupsthat are J coupled. The "fingerprint" region contains (¹ H^(N), ¹H.sup.α) cross-peaks from the peptide backbone. The degree of resolutionof the "fingerprint" region of the COSY map obtained in H₂ O is a goodpredictor of the success of sequence-specific assignments to be obtainedwithout recourse to isotopic labeling. Transferred nuclear Overhausereffect (TRNOE) spectra (¹ H NMR) relies on different 2D NOE spectra,and, in essence, looks at the conformation of the ligand just after ithas dissociated from the protein. The use of TRNOE presumes, however,that the bound and free ligands are in fast exchange on the chemicalshift time scale, which translates to a ligand K_(D) greater than orequal to about 1×10⁻⁴ M. TRNOE methods are useful to crosscheck andaugment the distance information obtained by other approaches.

It is not intended that the present invention be limited by theparticular method used to obtain structural information. Furthermore, itis not intended that the present invention be limited to a search forany one type of drug; one or more of the molecules may benaturally-occurring or may be synthetic, or may be a chemically-modifiedform of a naturally-occurring molecule.

In some embodiments, it is desirable to compare the structure of hPAK65protein to the structure of other proteins. This will aid in theidentification of and selection of drugs that either selectively affecthPAK65 or have a broad-spectrum effect on more than one species ofrelated polypeptide (e.g., other rac1-related proteins). In oneembodiment of the invention an assay for determining hPAK65 proteinkinase activity is provided.

In another embodiment of the invention, an hPAK65 kinase inhibitionassay is provided, which assay can be used for screening drug librariesor agents for their capability to inhibit an hPAK65 kinase activity. Inone embodiment of this invention a method is provided for identifyingagents which inhibit hPAK65 kinase activity, said method comprisingadministering an agent to a reaction mixture containing substantiallypurified hPAK65, preferably activated hPAK65, a substantially purifiedsubstrate, e.g. MBP, and γ-³³ P-ATP, and then determining the extent towhich the agent inhibits phosphorylation of substrate as compared to acontrol reaction lacking the agent.

Accordingly, also provided is an assay kit for identifying agents whichinhibit hPAK65 kinase activity wherein the kits contain substantiallypurified polypeptide containing hPAK65 or more preferably constitutivelyactivated hPAK65 or a fragment thereof with constitutive activity.Activated hPAK65 can be provided in numerous forms including as theautophosphorylated form, as an analog having at least oneautophosphorylation-target serine replaced by an amino acid that mimicsphosphoserine, e.g. Glu or Asp (Huang and Erikson (1994) Proc. Natl.Acad. Sci. 91: 8960-8963), and as an N-terminal truncated form in whichthe hPAK65 regulatory domain responsible for inhibiting serine kinaseactivity is deleted. The assay kit can further contain a substantiallypurified substrate, such a MBP, and can further contain a bufferedaqueous solution and γ-³³ P-ATP. Preferred serine-substirutedconstitutively activated analog forms of hPAK65 are those in whicheither or both serines at amino acid position 381 or 383 are replaced byglutamic or aspartic acid. The threonine at position 384 may beoptionally replaced by glutamic or aspartic acid. Also preferred arehPAK65 having C-terminal mutations where the amino acids 489-491 (i.e.,Ser-Ser-Leu) are modified or removed. One embodiment of this type has adeletion of amino acids 482 to 506. A preferred embodiment has both thepreviously discussed mycepitope tag and the 482-506 deletion.

In one embodiment of the invention a method for inhibiting a hPAK65kinase is provided which includes the steps of contacting a compositioncontaining a hPAK65 kinase with an agent having the capability toinhibit hPAK65 kinase activity as determined in a hPAK65 kinaseinhibition assay described herein. Preferably the composition comprisesa body fluid of a mammal, more preferably the body fluid is blood or ablood fraction. Preferably PAK65 is a human PAK65. The method canfurther include the steps of measuring hPAK65 kinase activity in saidbody fluid in the presence and absence of said inhibitor and relatingsaid kinase activity to concentration of hPAK65 kinase or substrate forhPAK65 kinase in said composition. The contacting can occur in vivo.

In another embodiment of the invention, a pharmaceutical composition forthe control of PAK65 kinase dependent diseases in mammals is providedwhich includes an agent having the capability to inhibit PAK65 kinaseactivity as determined in a PAK65 kinase activity assay and apharmaceutically acceptable carrier.

In yet another embodiment of the invention is provided a method ofcontrolling a PAK65 kinase dependent disease, which includes the stepsof administering to a mammal suffering from a PAK65 kinase dependentdisease a PAK65 kinase dependent disease controlling amount of an agenthaving the capability to inhibit PAK65 kinase activity as determined ina PAK65 kinase inhibition activity assay.

A method for identifying compounds which inhibit PAK65 p21-bindingactivity is also provided. The method includes the steps ofadministering a compound in admixture with a substantially purified p21protein to a reaction mixture comprising substantially purified PAK65,and determining the extent to which the agent inhibits binding ascompared to a control reaction lacking the compound.

In another embodiment of the invention a method for inhibiting a hPAK65p21-binding activity is provided, which includes the steps of contactinga composition containing a hPAK65 with an agent having the capability toinhibit hPAK65 p21-binding activity as determined in a hPAK65p21-binding inhibition assay.

Accordingly, also provided is an assay kit for identifying agents whichinhibit or modulate hPAK65 p21-binding activity that includessubstantially purified hPAK65. The assay kit can optionally contain asubstantially purified p21 protein, preferably rac1 or CDC42Hs, abuffered aqueous solution, GTPγS, or an antibody to the Glu-Glu epitopetag attached to the N-terminal of rac1 or CDC42Hs. GTPγS is a form ofGTP that cannot be hydrolyzed to GDP by p21 GTPases.

A method for identifying agents which inhibit or modulate phosphaterelease from hPAK65-bound p21 proteins is provided. The method includesthe steps of contacting a substantially purified p21 protein with areaction mixture containing substantially purified hPAK65 to form a p21.protein-PAK65 complex, then contacting the complex with the test agent,and then determining the extent to which the agent inhibits phosphaterelease as compared to a control reaction lacking the agent. The hPAK65can be bound to a solid support or can be free in solution.

In another embodiment of the invention a method for inhibiting ormodulating phosphate release from hPAK65-bound p21 protein is provided,which includes the steps of contacting a composition containing ahPAK65-bound p21 protein with an agent having the capability to inhibitor modulate phosphate release from hPAK65-bound p21 protein asdetermined in a phosphate release assay.

Accordingly, also provided is an assay kit for identifying agents whichinhibit or modulate such phosphate release. The kit includessubstantially purified hPAK65. The assay kit can optionally contain asubstantially purified p21 protein, preferably rac1 or CDC42Hs, abuffered aqueous solution, and GTPγP³².

The invention now provides for the first time the ability to identifycellular substrates for hPAK65 kinase activity that are involved inhPAK65-related pathways. Appropriate use of the protein and nucleic acidhPAK65 embodiments provided by this invention, preferably with thetissue sources or cell lines indicated herein as expressing hPAK65, andparticularly constitutively activated hPAK65, will allow identificationof those cellular macromolecules that are phosphorylated when hPAK65serine kinase activity is activated.

The agents identified by the various embodiments of this invention areall readily adapted to therapeutic use as hPAK65 kinase inhibitors (oras hPAK65-rac1 /CDC42Hs binding and/or autophosphorylation inhibitors)for the control of PAK65 kinase dependent diseases in mammals. PAK65kinase dependent diseases can include hyperproliferative disorders whichare initiated/maintained by aberrant PAK65 serine kinase enzymeactivity. Examples include cancer, atherosclerosis, and antiangiogenesis(e.g., tumor growth, diabetic retinopathy). It is understood in the artthat therapeutically useful kinase inhibiting agents preferably shouldbe selective. PAK65 kinase inhibitors that inhibit many other proteinkinases as a result of their lack of specificity are highly cytotoxic.Therefore, routine assays which measure cytotoxicity can be usedidentify PAK65 inhibitors which are likely to produce undesired sideeffects due to a lack of selectivity. As a more detailed test ofselectivity, compounds should be tested for their ability to inhibit theenzymatic activity of a range of other protein kinases, e.g. classes ofprotein kinases identified based upon the amino acid(s) that serves astheir substrate: kinases that phosphorylate tyrosine, kinases thatphosphorylate tyrosine and threonine, and kinases that phosphorylateserine and threonine. Examples of kinases that phosphorylate serine andthreonine include RAF, protein kinase A, protein kinase C, and TGF betareceptor. The kinase MEK is an example of kinases that phosphorylatetyrosine and threonine.

In the following discussion of uses of kinase inhibitors, the discussionfocuses on hPAK65 protein kinase, however, it should be understood thatany discussion here of use of a compound as a hPAK65 kinase inhibitor isgenerally applicable to use of an agent that is specific for one of theother activities of hPAK65. Whether an agent is specific for hPAK65kinase activity is readily determined by use of the kinase activityassays set out in the examples.

In order for compounds that inhibit (or modulate) hPAK65 kinase activityor one of its other activities to be therapeutically useful they shouldbe active on intact cells. Several methods are readily available fordetermining the activity of candidate hPAK65 inhibitors (or modulators)against hPAK65 on intact cells. Phosphorylation of the hPAK65 cellularsubstrates can be measured using antiphosphoserine antibodies orphosphopeptide fingerprints. Also, additional intracellular signalingevents can be measured including calcium flux, inositol phosphatemetabolism, cellular proliferation, and cellular DNA synthesis, or anyof the other physiological process related to hPAK65 dependent pathways.Preferably, a cell line genetically engineered to express constitutivelyactive hPAK65 may lead to a transformed cell line upon which candidatehPAK65 kinase activity inhibitors can be tested for the ability todecrease hPAK65 kinase activity, decrease overall phosphorylation in acell, or reverting the cell to a less transformed state.

Candidate agents that inhibit or modulate p21 protein binding to hPAK65can be further tested against a cell line transformed by a recombinantrac 1 that has been altered into a constitutively active form. Therac1-transformed cells have the characteristics of neoplastic cells,e.g. of forming loci and tumors in mice, and can be analyzed forreversion to a more normal state in the presence of candidate agentsthat inhibit rac1 activation of hPAK65.

It is likely that solubility of the agents of the present invention bothin water and in mildly hydrophobic solvents will enhance the probabilitythat they traverse the cell membrane.

Compounds of this invention may be useful in the form of the free acid,in the form of a salt and as a hydrate. All forms are within the scopeof the invention. Basic salts may be formed and are simply a moreconvenient form for use; in practice, use of the salt form inherentlyamounts to use of the acid form. The bases which can be used to preparethe salts include preferably those which produce, when combined with thefree acid, pharmaceutically acceptable salts, that is, salts whoseanions are non-toxic to the animal organism in pharmaceutical closes ofthe salts, so that the beneficial properties inherent in the free acidare not vitiated by side effects ascribable to the cations. Althoughpharmaceutically acceptable salts of the acid compound are preferred,all salts are useful as sources of the free acid form even if theparticular salt per se is desired only as an intermediate product as,for example, when the salt is formed only for purposes of purificationand identification, or when it is used as an intermediate in preparing apharmaceutically acceptable salt by ion exchange procedures.

Agents within the scope of this invention that have activity as specificinhibitors or modulators of hPAK65 possess therapeutic value as cellularantiproliferative agents for the treatment of certain conditionsincluding, for example, neoplasia, inflammation, lymphoproliferativeconditions, arthritis, autoimmune diseases, apoptosis, and the like.

Compounds of the present invention can be administered to a mammalianhost in a variety of forms i.e., they may be combined with variouspharmaceutically acceptable inert carriers in the form of tablets,capsules, lozenges, troches, hard candies, powders, sprays, elixirs,syrups, injectable or eye drop solutions, and the like depending on thechosen route of administration, e.g., orally or parenterally. Parenteraladministration in this respect includes administration by the followingroutes: intravenous, intramuscular, subcutaneous, intraocular,intrasynovial, transepithelial (including transdermal, ophthalmic,sublingual and buccal), topical (including ophthalmic, dermal, ocular,rectal, nasal inhalation via insulation and aerosol), and rectalsystemic.

The active compound may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsules, or it may be compressedinto tablets, or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipient and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 6% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 1 and 1000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as polyvinylpyrrolidone, gum tragacanth,acacia, sucrose, corn starch or gelatin; an excipient such as calciumphosphate, sodium citrate and calcium carbonate; a disintegrating agentsuch as corn starch, potato starch, tapioca starch, certain complexsilicates, alginic acid and the like; a lubricant such as sodium laurylsulfate, talc and magnesium stearate; a sweetening agent such assucrose, lactose or saccharin; or a flavoring agent such as peppermint,oil of wintergreen or cherry flavoring. Solid compositions of a similartype are also employed as fillers in soft and hard-filled gelatincapsules; preferred materials in this connection also include lactose ormilk sugar as well as high molecular weight polyethylene glycols. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, flavoring such as cherry or orange flavor,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof. Of course, any material used in preparing any dosage unit formshould be pharmaceutically pure and substantially non-toxic in theamounts employed. In addition, the active compound may be incorporatedinto sustained-release preparations and formulations.

The active compound may also be administered parenterally orintraperitoneally. For purposes of parenteral administration, solutionsin sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions of the corresponding water-soluble,alkali metal or alkaline-earth metal salts previously enumerated. Suchaqueous solutions should be suitable buffered, if necessary, and theliquid diluent first rendered isotonic with sufficient saline orglucose. Solutions of the active compound as a free base or apharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant such as hydroxypropylcellulose. A dispersion canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal injection purposes. In this connection, the sterileaqueous media employed are all readily obtainable by standard techniqueswell-known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze drying techniquewhich yield a powder of the active ingredient plus any additionaldesired ingredient from the previously sterile-filtered solutionthereof.

For purposes of topical administration, dilute sterile, aqueoussolutions (usually in about 0.1% to 5% concentration), otherwise similarto the above parenteral solutions, are prepared in containers suitablefor drop-wise administration to the eye. The therapeutic compounds ofthis invention may be administered to a mammal alone or in combinationwith pharmaceutically acceptable carriers. As noted above, the relativeproportions of active ingredient and carrier are determined by thesolubility and chemical nature of the compound, chosen route ofadministration and standard pharmaceutical practice. The dosage of thepresent therapeutic agents which will be most suitable for prophylaxisor treatment will vary with the form of administration, the particularcompound chosen and the physiological characteristics of the particularpatient under treatment. Generally, small dosages will be used initiallyand if necessary, will be increased by small increments until theoptimum effect under the circumstances is reached. Oral administrationrequires higher dosages. The compounds are administered either orally orparenterally, or topically as eye drops. Dosages can be readilydetermined by physicians using methods known in the art, using dosagestypically determined from animal studies as starting points.

The invention now being generally described, the same will be betterunderstood by reference to the following detailed examples, which areprovided for the purpose of illustration only and are not to beconsidered limiting of the invention unless otherwise specified.

EXAMPLES Example 1

Identification of Effector Proteins in Neutrophils for rac1 and CDC42Hs

In order to detect whether targets for rac/CDC42Hs exist in humans, anoverlay assay was used to detect any such targets in neutrophil cytosol.The overlay assay for GTPase was initially described as a method todetect GTPase activating proteins ("GAP") (Manser et al. 1992, which isincorporated herein by reference) for rho-like proteins; by probing anitrocellulose filter containing lysate with (γ³⁵ p)GTP bound rho-likeproteins it was possible to detect proteins on the filter which canaffect the GTP hydrolysis rate, since GAPs catalyze the release of theγPi from GTP, the loss of radiolabeled Pi from the GTPase probe wasvisualized as clear bands over a dark background. However, by modifyingthe probing conditions it was discovered by the present inventors thatone can detect effector proteins that inhibit the release of Pi as darkbands on the blot. As disclosed herein three major bands clustering nearthe 68 Kda marker were detected using human neutrophil cytosol. 40 μl ofcrude or partially purified neutrophil cytosol fraction containing p65(˜80 μg protein) were applied on a 14% SDS PAGE and blotted to a PVDFmembrane. The membrane was stained for 30 sec with Coomassie Blue stainto detect transferred proteins, destained for 2 min., and incubated for30 min with PBS containing 1% BSA, 0.5 mM MgCl₂ 0.1% Triton x-100 and 5mM DTT. 30-50 μl (3-5 μg protein) of either CDC42Hs, human rac1, orhuman rho A, prepared as recombinant proteins in insect Sf9 cells, werediluted into 200 μl exchange buffer: 25 mM MES, pH 6.5, 50 mM NaCl, 5 mMEDTA, 0.05% Triton x-100 and 1 μl of [γ³² p]GTP (5 μCi, ICN) or [β³²p]GDP (5 μCi, ICN). The proteins were incubated with the exchange bufferfor 15 min at room temperature and then were mixed with 10 ml of bindingbuffer containing 25 mM MES buffer pH 6.5, 0.5 mM GTP, 5 mM MgCl₂, 50 mMNaCl, and 5 mM DTT. Immediately, thereafter the nucleotide-loadedprotein was used to probe the filter. The mixture was incubated for 5-8min and washed for five min. with 25 mM MES buffer, pH 6.5, 5mM MgCl₂,0.05% Triton X-100. The membrane was dried and exposed to a film for 2-3hours. Three major proteins were detected as targets for rac1 /CDC42Hs.It was further determined that these proteins were abundant in cytosolicfractions of neutrophils and HL-60 cells. Accordingly, neutrophils wereused as a source for purifying the protein. Fractionation of neutrophilcytosol on a Mono Q column resolved the bands clustering near the 68 kDamarker into three distinct bands of molecular size 62, 65 and 68 kDa,all of which were detected only when the filter was probed with theGTP-bound form of rac1 and CDC42Hs and not with GTP-bound rho A. SeeFIGS. 1 and 3.

Example 2

Isolation of hPAK65 Protein

The p65 band, which was the most abundant of the three proteinsidentified in Example 1, was subsequently isolated and purified. Cytosolwas prepared from human neutrophils as previously described (Abo et al.1994, which is incorporated by reference). All purification steps wereperformed on columns connected to a FPLC system, at 4⁰ ° C., flow rateof 1 ml/min, and 1 ml fractions were collected. 10 ml (10 mg/ml) ofneutrophil cytosol were applied on a Mono Q column (HR5/5 Pharmacia LKB)equilibrated with buffer A: 20 mM Tris-HCl, pH 7.4, 1 mM DTT 5 mM MgCl₂,1 mM PMSF, 1 μg /ml pepstatin. The proteins were eluted with a 30 mlgradient from 0 to 0.5 M NaCl and the collected fractions were assayedfor rac1 or CDC42Hs binding by the overlay assay. Fractions containingthe p65 protein were pooled and subjected to ammonium sulfateprecipitation. Ammonium sulfate grains were added to the mixture over aperiod of 15 min to achieve 40% saturation. The solution was stirred onice for an additional 30 min., and subsequently centrifuged at 100,000 gfor 15 min in a TLX Beckman ultracentrifuge. The pellet was resuspendedin buffer A to its original volume and the fractions were analyzed bythe overlay assay. The supernatant obtained by 40% ammonium sulfatesedimentation which contained the desired p65 protein was furtherpurified on a Phenyl Superose column (HR 5/5 Pharmacia LKB) equilibratedwith 100 mM Pi buffer, pH 7.2, 1 mM DTT, 5 mM MgCl₂, 1 mM PMSF, 1 μg/mlpepstatin and 1.2 M ammonium sulfate. The bound proteins were eluted by30 ml gradient from 1.2 M to 0 ammonium sulfate. In the case of thepurification of a large amount of starting material (300-500 mgprotein), the ammonium sulfate and the Phenyl Superose steps were thefirst purification procedures followed by the Mono Q fractionation. Forthis purpose a Hiload Phenyl Sepharose 16/10 column (Pharmacia, LKB) wasused. Collected fractions were analyzed by the overlay assay andfractions containing the p65 protein were pooled and desalted intobuffer A on a PD 10 column (Pharmacia, LKB). The partially purified p65was further purified on a Mono S column HR5/5 (Pharmacia LKB)equilibrated with the buffer A. Proteins were eluted by 30 ml gradient 0to 0.5 M NaCl and the fractions were analyzed by the overlay assay. Atthis stage the p65 polypeptide could be easily identified by CoomassieBlue staining and the band was excised and used for amino acid analysis.

Amino acid sequence was determined as follows. The p65 preparation waspurified by SDS-PAGE, following staining with Coomassie Blue G-250, andthe protein was excised. After washing, gel pieces were macerated anddigested with Achromobacter lyticus endoproteinase Ly-C. Peptides wererecovered by sequential washes and separated by tandem hplc using 2.1 mminternal diameter anion exchange and reverse phase columns in series,following previously described procedures (Kawasaki et al. 1990).Fractions were collected and applied directly to an Applied Biosystem477A pulsed liquid automated sequencer modified for fast cycle chemistryas described (Totty et al. 1992). Digestion of p65 and subsequent aminoacid analysis yielded the following amino acid sequence:STMVGTPYWMAPEVVTR, which is closely related to a sequence within theserine/threonine kinase domain of yeast STE20 (Ramer and Davis 1993),and 100% identical to a rat brain serine/threonine kinase, PAK65 (Manseret al. 1994).

Example 3

Isolation of hPAK65 cDNA Clone

Based on the protein sequence of rat brain PAK65 (Manser et al. 1994)and the amino acid sequence derived from purified p65 determined inExample 2, the oligoimers GM749 5' GGGGCCATCCAATAGGGGGTACCNACCATNG 3'and GM752: 5' ACCGGAGAATTCACCGGCATGCCTGAACAGTGG 3' were designed andused to amplify human cDNAs encoding PAK proteins. These oligomers wereused to amplify specific PAK cDNAs from several commercially availablehuman cDNA libraries. Although the expected product was detected inseveral tissue specific libraries, relatively large amounts weredetected in a human placenta library. A human placenta library fromStratagene (1994 catalog number 936203) was selected as a source for acDNA clone. The gel-purified 962 base pair PCR product was amplified inthe presence of ³² P dCTP and ³² P dGTP resulting in a radioactiveproduct. This PCR product was used to screen approximately 100,000recombinant plaques using stringent hybridization conditions(hybridization buffer: 50% formamide, 5×SSC, 5×Denhardt's, 50 mM NAPO₄pH7, and 0.1% SDS at 42° C.; wash: 5×SSC, 0.1% SDS) in order to isolatea full-length cDNA. One positive clone was plaque purified andautoexcised ("in vivo excision") from lambda Zap II according to themanufacturer's protocols to yield the plasmid clone containing the fulllength human PAK65 cDNA with the sequence presented in FIG. 2A (SEQ IDNO: 1), which was designated pBSPAK. The sequence of the cDNA insertcontained within the resulting Bluescript plasmid was determined usingthe dideoxynucleotide chain termination method.

Example 4

Amino Acid Homology

The nucleotide and deduced amino acid sequence of the human PAK65 cDNA,which will be referred to here as hPAK65, is shown in FIG. 2. A fulllength hPAK65 cDNA clone obtained from a human placental librarydisplayed sequence similarity to the kinase domain of rat brain PAK65and yeast STE20. Although both rat brain PAK65 and hPAK65 exhibitsimilar specificity for rac1/CDC42Hs the sequences display only 70%sequence identity at the amino acid level in the rac1/CDC42Hs bindingdomains. The complete amino acid sequence of hPAK65 shares ˜73% identityto the previously isolated PAK65 from rat brain (Manser et al. 1994)(FIG. 2A) and shares more then 95% identity within the kinase domain(amino acids:230-506) (FIG. 2B). In addition, hPAK65 exhibits ˜63%identity to the kinase domain of STE20 (position:620-880) (Ramer andDavis 1993) (FIG. 2C). As reported for rat PAK65 (Manser et al. 1994),the rac1 and CDC42Hs binding domain (amino acid 47-113) of hPAK65 alsoshare some similarities to the STE20 regulatory domain. (data notshown).

Example 5

Tissue Distribution of hPAK65

To determine tissue distribution of mRNAs encoding hPAK65 a radioactivePCR product containing the highly conserved kinase domain (base pairs1009 through 1912) of human PAK cDNA was used to hybridize MRNA derivedfrom 16 human tissues and 8 cancer cell lines immobilized on Northernblots (membrane-bound mRNA is available from Clontech). Since the probewas derived from the kinase domain, the most conserved region amongsthPAK65, rat brain PAK65, and STE20, it was expected that the expressionof closely related messages would be analyzed. The hybridizationconditions used were as suggested by the manufacturer using ExpressHyb(Clontech) except that the temperature was 72° C. and hybridization timewas overnight. The northern blot analysis indicates that hPAK65 relatedmRNA is ubiquitously distributed among various tissues with higherlevels in cells of myeloid origin, namely in skeletal muscle, ovary,thymus, and spleen and 2-3 fold higher in HL-60 cell line (FIG. 3). FourRNA species were detected in most of the tissues with sizes ofapproximately 7.5 kb, 5 kb, 4.4 kb and 3 kb. The 7.5 kb message was thepredominant species in all the tissues except skeletal muscle where the7.5 kb and the 3 kb mRNA were roughly the same. In contrast, the celllines HL-60, Molt-4, Raji and SW480 have equal amounts of all fourspecies whereas, in brain a different size mRNA was detected around 3.3kb. Based on results of genomic Southern blotting under stringentconditions (results not shown; membrane-bound human genomic DNAavailable from Clontech; conditions were that as suggested by themanufacturer except the hybridization temperature was 64° C. rather than60° C. and hybridization went overnight), these multiple mRNA are mostlikely alternatively spliced forms from a single gene (FIG. 3).

In contrast, by the use of the overlay assay, a high level of rat PAKproteins was detected mainly in brain cells (Manser et al. 1994),whereas by northern analysis higher expression of hPAK65 in neutrophilsand HL-60 cells was found as described herein. It is most likely thatthe overlay assay is not sensitive enough to detect PAK in tissues withrelatively lower expression of protein, and hence is not a preferredmethod for determining the tissue distribution of PAK.

Example 6

Production of Recombinant Proteins in Sf9 Cells

A modified Glu-Glu epitope tag (Grussenmeyer et al. 1985; modified toMet-Glu-Tyr-Nlet-Pro-Thr-Asp) was cloned onto the N-terminus of rac1,CDC42Hs, and rho using a polymerase chain reaction (Grussenmeyer et al.1985). hPAK65 was myc-epitope tagged (MEQKLISEEDL) by ligating annealedoligomers into the Xba I site (417 bp) immediately upstream of theinitiation Met of hPAK65. The tagged cDNAs were cloned into thebaculovirus expression vector pAcC13. pAcPAK780 contains the myc-taggedhPAK65. 1 g of snap frozen Sf9 pellets, expressing the desired proteins,were Dounce homogenized in 10 ml of Buffer B: 50 mM Tris-HCl, pH 7.5,150 mM NaCl, 5 mM MgCl₂ 200 μM GDP, 1 mM Pefabloc, 10 μg/ml leupeptin,10 μg/ml aprotinin. To remove the particulate fraction, the homogenatewas centrifuged at 100,000 g for 15 min. The soluble fraction wasapplied to 2 ml protein G Sepharose column conjugated either withanti-Glu-Glu or anti-Myc monoclonal antibodies. The column was washedwith 10 ml of buffer B lacking GDP, and the protein was eluted with thesame buffer containing either the Myc peptide tag or 50 μg/ml of the EDpeptide tag (EYMPTD). Fractions were analyzed on a SDS-PAGE, quantitatedby the Bradford method, concentrated by a Centricon 10 (Amicon) to 1mg/ml, aliquoted, snap frozen and stored at -70° C. A fresh aliquot ofthe protein was used for each assay. Expression of the hPAK65 in Sf9cells allowed preparation of essentially homogeneous recombinantmyc-epitope tagged hPAK65 ("rhPAK65"), which was used forcharacterization the biochemical properties of hPAK65.

Example 7

The Binding Specificity of hPAK 65 to p21 Proteins

Recombinant hPAK65 or the endogenous hPAK65 in neutrophil cytosol, wasdetected only when the filter was probed with [γ³² P]GTP-CDC42Hs and[γ³² P]GTP-rac1 but not with [γ³² P]GTP-rho A (FIG. 4). No proteins weredetected when the GTPase was preloaded with [β³² P]GDP, indicating thatthe hPAK65 protein behaves as an effector molecule for rac1 and CDC42Hs.The relative affinity as judged by the overlay assay is ˜3-4 fold higherfor CDC42hs than for rac 1 (FIG. 4).

Example 8

CDC42Hs and rac1 Induce Autophosphorylation of hPAK65.

To determine whether CDC42Hs and rac 1 would induce autophosphorylationof hPAK65, hPAK65 was incubated with the activated form of either rac1or CDC42Hs in a kinase reaction containing [γ³² P]ATP. Stimulation ofautophosphorylation of hPAK65 was observed in both cases (FIG. 5A). Nophosphorylation was observed with rho A or by omitting the GTPase andsimply adding GTP (FIG. 5A). Phosphorylation occurred in a dosedependent manner only with the GTP or GTPγS form of CDC42Hs or rac1.Maximal phosphorylation was obtained after 15 min at 30° C. (data notshown).

To determine the phosphorylation pattern on hPAK65 induced by rac 1 andCDC42Hs, hPAK was subjected to kinase reaction containing eitheractivated rac1 or CDC42Hs. To remove rac1 and CDC42Hs, the hPAK65immobilized on beads was washed three times with PBS containing 1%Triton X-100 and the beads were resuspended in 100 mM Tris HCl, pH 6.8,0.5% SDS, 10 mM DTT, 10% glycerol. The samples were boiled for 3 min and10 μg of the indicated protease (all from Boehringer) were added . Theproteins were digested overnight at room temperature and the sampleswere analyzed on 16% Tricine gels. The gel was stained with CoomassieBlue, destained, dried and exposed to film overnight. Phosphoamino acidanalysis was performed to determine the location of autophosphorylation.4 μg (on 50 μl beads) of recombinant hPAK65 were subjected to kinasereaction as described above. The phosphorylated hPAK65 immobilized onSepharose G beads were washed three time in PBS containing 1% TritonX-100 and then hydrolyzed in 50 μl of 6 N HCl at 100° C. for 2 hrs. Thebeads were removed by centrifugation, and the supernatant was dried anddissolved in 10 μl of pH 3.5 electrophoresis buffer (10:100:1890.pyridine:acetic acid:water). Phosphoamino acids were resolved on a thinlayer cellulose plate using the electrophoresis buffer essentially asdescribed before (Cooper et al. 1983). Standards were visualized bystaining with 0.2% Ninhydrin in acetone and ³² Pi Labeled residues weredetected by autoradiography overnight. Phosphoamino acid analysisindicated that hPAK65 was phosphorylated on serine residues whenactivated by CDC42Hs and not on threonine or tyrosine residues (FIG.5B).

Since rac1 and CDC42Hs share ˜72% sequence identity and apparently playdifferent. physiological roles, it is conceivable that rac1 and CDC42Hsmay activate hPAK65 autophosphorylation on distinct sites. To test thishypothesis hPAK65 was incubated either with rac1 or CDC42Hs in a kinasereaction with [γ³² P]ATP and the phosphorylated protein was digestedwith three different enzymes: trypsin, chymotrypsin and Glu-C. Thistreatment resulted in the generation of radiolabeled phosphopeptideswhich were resolved on 16% a Tricine gel Identical phosphopeptideprofiles were generated from digestion of hPAK65 activated by either racor CDC42Hs (FIG. 5C). This result suggest that rac and CDC42Hs stimulatehPAK65 to phosphorylate itself on the same serine sites.

Like human neutrophil p65 and rat brain PAK65, recombinant hPAK65interacted specifically with the activated form of either rac1 or CDC42,and subsequently, if provided with ATP, become autophosphorylated. Thedata suggest that rac/CDC42Hs mediates hPAK65 autophosphorylationthereby generating an active kinase. The strict requirement for hPAK65binding and activation by only the GTP-bound form of rac and CDC42Hsindicates that hPAK65 serves as an effector protein for rac/CDC42Hs. Inaddition, it is most likely, that the relative binding affinity ofrac/CDC42Hs for hPAK65 is regulated by their nucleotide state and not bythe phosphorylated state of hPAK65. In contrast to the model suggested(Manser et al. 1994), the data disclosed by the present inventorsindicates that the phosphorylation state of hPAK65 is not involved inregulating rac/CDC42Hs binding. Phosphorylated and unphosphorylatedhPAK65 exhibited comparable affinities for rac1 and CDC42Hs.

Example 9

Phosphorylated hPAK is an Active Kinase

A kinase assay was performed in order to determine whether hPAK65 hadkinase activity towards other proteins and whether that activity wasaffected by p21 proteins. 1-2 μg of rhPAK65 (bound to protein GSepharose conjugated with monoclonal Myc antibody) were washed once andincubated in 40 μl of kinase buffer (50 mM Tris-HCl, ph 7.5, 100 mMNaCl, 10 mM MgCl₂, 1 mM MnCl₂) with 1-2 μg of either rac1, rho, orCDC42Hs, which were all previously loaded with GTP or GDP. The reactionwas initiated by adding 10 μl of kinase buffer containing 50 μM ATP and5 μCi [γ³² P]ATP, and incubated for 20 min at 30° C. The reaction wasstopped by adding 10 μl of 5×SDS PAGE sample buffer, and boiling for 5min. Samples were applied to a 14% SDS PAGE, the gel was stained withCoomassie Blue, destained, dried and exposed to a film for 1-2 hrs.Phosphorylated bands were excised and the incorporated ³² phosphateswere counted. In the case of phosphorylation of myelin basic protein("MBP"), 3 μg of MBP (Sigma) was included in the kinase reaction.

Phosphorylation of hPAK65 induced by rac1 or CDC42Hs stimulates itskinase activity towards MBP substrate (FIG. 6a). Both rac and CDC42Hswere able to stimulate an active hPAK65 kinase in a time dependentmanner. Maximal MBP phosphorylation was obtained within 10 min at 30° C.(FIG. 6B).

Example 10

Activated hPAK Does Not Require rac1 or CDC42Hs to Sustain its KinaseActivity

The above experiments clearly demonstrate the requirement forrac1/CDC42Hs in the activation of hPAK65 autophosphorylation. Whetherrac1/CDC42Hs are required for hPAK65 kinase activity was investigated by-the following experiment: hPAK65 autophosphorylation was first inducedby CDC42Hs in the presence of cold ATP, then hPAK65 and CDC42Hs complexwas disrupted by exhaustive washes. The autophosphorylated hPAK65 freeof CDC42Hs (confirmed by western blot) was subjected to a second kinasereaction containing [γ³² P]ATP and MBP. Autophosphorylated hPAK65 freeof CDC42Hs was sufficient to activate MBP phosphorylation (FIG. 6C). Thecontrol hPAK65 was treated exactly the same except that ATP was notincluded in the first kinase reaction. Lower levels of hPAK65autophosphorylation were detected on the control hPAK65, which mostlikely are due to residual levels of unwashed CDC42Hs/PAK complexes.These data suggest that rac and CDC42Hs play an important role in theactivation of hPAK65 by stimulating its autophosphorylation but not inthe maintenance and regulation of the kinase activity.

Example 11

Comparison of the Binding of CDC42Hs to the Phosphorylated vs.Unphosphorylated hPAK65.

Fully phosphorylated and unphosphorylated hPAK65, as judged by mobilityshift and kinase reaction, bound equally well to activated CDC42Hs.(FIG. 7) Phosphorylation of hPAK65 most likely serves to activate thekinase of hPAK65 but does not to alter its affinity for rac/CDC42Hs.This result is in contrast to the rat system, wherein it was shown thatCDC42 has a reduced affinity for the activated form of rat brain PAK65and was suggested that phosphorylation of PAK is a mechanism by whichrac/CDC42 can be released from PAK once activated (Manser et al. 1994).

Example 12

The Effect of hPAK65 on Intrinsic and Stimulated GTPase Activity

Since the p65 protein was initially detected by the overlay assay as apotential effector and GTPase inhibitor for rac1/CDC42Hs, the effect ofhPAK65 on GTPase activity in solution was examined. Purified CDC42Hs(800 nM) was prebound to 80 nM g-32P-GTP (6000 Ci/mmol) in the presenceof 1 mM EDTA for 5 min at 25° C., followed by addition of 19 volumes GAPAssay Buffer (50 mM MES, pH 6, 100 mM NaCl, 5 mM MgCl₂) to yield 4 nMγ-32P-GTP-CDC42Hs. 1 nM [γ-³² P]-GTP-CDC42Hs was incubated with theindicated concentrations of hPAK65, which had been preincubated in thepresence or absence of ATP. Reactions were carried out in GAP AssayBuffer containing BSA (0.2 mg/ml BSA, 50 mM MES, pH 6.5, 100 mM NaCl, 5mM MgCl₂) in the presence or absence of 20 nM human p190-catalyticfragment for 5-10 min at 25° C., followed by assay for phosphate release(Shacter 1984). For p190-stimulated reactions, corresponding reactionsin the absence of p190 were performed and the resulting hydrolysissubtracted to yield only p190-dependent activity. p190 is a stimulatorof GTPase activity.

hPAK65 does exhibit a marginal effect on CDC42Hs intrinsic GTPaseactivity (FIG. 8A). Increasing the amount of hPAK65 up to 1000 fold overCDC42Hs inribited the intrinsic GTPase activity but only by 10-15% (FIG.8A). Interestingly, activated hPAK65 had an identical effect, suggestingthat phosphorylation has no regulatory effect on rac/CDC42Hs intrinsicGTPase activity (FIG. 8A).

In contrast, when the catalytic domain of human p190 GAP (Settleman etal. 1992 disclosed rat p190) was included in the assay, hPAK65 exerted asignificant inhibition of the GTP hydrolysis stimulated by p190.Increasing the concentration of hPAK65 to 5 fold greater than that ofCDC42Hs resulted in blocking up to 40% of the GAP stimulated GTPhydrolysis (FIG. 8B). No differences in GAP stimulating GTPase wereobserved when phosphorylated HPAK 65 was compared to theunphosphorylated (FIG. 8B).

Rac1 and CDC42Hs share ˜72% sequence identity and have distinctphysiological roles. For instance, rac1 induces membrane ruffling(Ridley et al. 1992) and interacts with p67-phox to activate the NADPHoxidase (Diekmann et al. 1994), whereas CDC42Hs had no effect on NADPHoxidase activity or on the induction of membrane rufflings. In yeast,CDC42 is involved in bud formation (Johnson and Pringle 1990). Therelative lower affinity of hPAK65 for rac1 compared to CDC42Hs maysuggest that CDC42Hs is the physiological activator of hPAK65. However,It is unlikely that hPAK65 activation by rac1 is an in vitro artifact,since in vitro activation of the NADPH oxidase specifically requires racand not CDC42Hs. It is most probable that rho-like proteins havenumerous effector domains, some of which may be shared among the variousfamily members, whereas others may be unique to each member. Since thepartial phosphopeptide map generated from activated hPAK65 either by racor CDC42Hs were identical, this indicates that rac and CDC42Hs activatehPAK65 in the same fashion; namely, by the stimulation theautophosphorylation at the same sites. It will be very important todetermine which agonist will link rac 1 or CDC42Hs stimulation withhPAK65 activation in vivo. Such studies will determine which nucleotideexchange factor for rho-like proteins is implicated in this pathway. Forexample, Dbl was shown to have a nucleotide exchange activity on rho,CDC42Hs but not on rac1 (Hart et al. 1991). Thus it is possible thatactivation of Dbl may lead to CDC42Hs-dependent hPAK65 stimulation.

The homology of hPAK65 and rat brain PAK65 to the kinase domain of yeastSTE20 suggests a role of rac/CDC42Hs and PAK proteins in the mitogenactiviated protein ("MAP") kinase cascade in mammalian cells (Avruch etal 1994) which is involved in cell proliferation and transformation.STE20 was shown to be a target for the βγ subunits of the hetrotrimericG protein in S. cerevisiae which link the pheromone response to a kinasecascade leading to transcription activation (Leberer et al. 1992, Erredeand Levin 1993). Five protein kinases (STE20, STE11, STE7, FUS3 andKSS1) were implicated between the G proteins and the transcriptionfactor STE12 (Errede and Levine 1993). STE7 has some homology to MAPkinase and FUS3 and KSS1 are yeast homologues of MAP kinase (Errede etal. 1993). In addition, genetic evidence demonstrates a functionalassociation between a novel gene STE5 and STE20 (Leberer et al. 1993).Besides a limited homology to FAR1, STE5 has no specific knownstructural motif however operates within this kinase cascade and is mostlikely functions as an adaptor protein (Leberer et al. 1993, Errede andLevine 1993). The relatively high divergence between brain rat PAK65 andhPAK65 in their regulatory domains, suggest that these proteins may becontrolled by different molecules. Thus, different mammalian STE5homologues may serve as adaptor proteins to assemble distinct PAK likekinases with downstream kinases such as STE7 and STE11. Rac is believedinvolved in activating stress kinase pathways that can lead to, amongother thigs, apoptosis. MEK kinase (MEKK), implicated in stress kinasepathways, may act downstream form hPAK65 since MEKK has homology toyeast STE11 which acts downstream from yeast STE20, a protein shownherein to have homology to hPAK65.

In summary the data presented herein are consistence with the modelpresented in FIG. 9. Upon exchange of the nucleotide from GDP to theactive form GTP, rac1/CDC42Hs interact with hPAK65 and subsequentlyinduce hPAK65 autophosphorylation. The GTP is hydrolyzed to GDP by theintrinsic GTPase activity of rac1/CDC42Hs. The inactive GDP-bound formof rac1/CDC42Hs are released from the active hPAK65 kinase whichsubsequently phosphorylates as yet unidentified physiological substrate.

Example 13

Antibodies to hPAK65

Antibody generated against a peptide derived from the kinase domain ofhPAK65 was used to determine whether the other two effector proteins,p62 and p68, identified in neutrophil cytosol are related to hPAK65proteins. No cross reactivity was observed using polyclonal sera.

Example 14

A rac/CDC42 hPAK65 Binding ELISA

The following ELISA format assay provides one embodiment of a means todetect or measure the binding of p21 proteins to hPAK65 or fragmentsthereof, and thus provide one means to screen for agents that modulatethis binding. A surface, eg. of a microtiter well, was coated withhPAK65 protein solution prepared by first adding 0.5 mM DTT toDulbecco's phosphate buffered saline (containing Ca²⁺ and Mg2+) followedby adding substantially purified GST-pakette fusion protein to a finalconcentration of 10 μg/ml (for 0.5 μg/well). After gentle mixingpreferably by inversion, 50 μl of this GST-PAKette solution was placedinto each well of a microtiter plate, e.g. Immulon plate, which was thencovered with plate sealer and incubated overnite, preferably at 4° C.and preferably with rocking to evenly distribute the solution across thesurface. Coated plates were typically useable for 4-5 days. Prior to usethe plate was washed once with 200 ul of wash buffer: PBS containingCa²⁺ and Mg²⁺ plus 0.1% tween-20. Non-specific binding sites wereblocked by adding to each well 200 ul of PBS containing Ca²⁺, Mg^(2+and)1% BSA, with incubation 4° C. for at least 2 hours, preferably withrocking, followed by two washes with 200 ul wash buffer each time.

Either recombinant tagged human rac1 or CDC42Hs were mixed at desiredconcentrations with 1 mM GDP/GTPγS and EDTA (at twice the MgCl₂ molarconcentration in order to chelate Mg²⁺) to a total of ˜3× the volume ofrac1/CDC42Hs added. The volume was adjusted with pre-bind buffer (50 mMTris pH 8, 50 mM NaCl). The p21 protein solution was then incubated at30° C. for 30 minutes. MgCl₂ was added to twice the EDTA molarconcentration. Magnesium ion stimulates nucleotide binding to p21proteins. EDTA chelates Mg²⁺ to aid in nucleotide removal.

For binding, the above p21 protein solution was adjusted to volume withbinding buffer (50 mM Tris pH 8, 50 mM NaCl, 50 mM MgCl₂, 1% NP-40 0.1%BSA). Binding was commenced by adding 50 ul of rac1/CDC42Hs solution toeach well. To test an agent for its ability to inhibit or modulatebinding, a test agent was added at 10 uM to the 50 ul solution (e.g., 5μl agent+45 μl rac1/CDC42 GTPγS solution). Binding was allowed toproceed for 1 hour at 35° C. The wells were then washed three times with200 ul wash buffer each time.

To detect hPAK65-bound rac1/CDC42Hs, an antibody-alkaline phosphataseconjugate ("Ab-AP") recognizing the appropriate tag was added to eachwell. Typically, antibody-AP conjugate was diluted 1:4000 into Abdilution buffer (PBS containing Ca²⁺ and Mg²⁺, 0.1% Tween-20, 0.1% BSA)and then 50 ul Ab-AP solution was added to each well. After incubationfor 1 hr at RT, each well was washed three times with 200 ul washbuffer. 100 μl of substrate for AP (stock: 1 tablet PNPP(Boeringher-Mannheim) in 100 mls H₂ O) was added to each well followedby incubation at 10-20 minutes at 35° C. to develop color. The color wasread at 405 nm when OD was about 1.0.

A candidate agent is one which significantly inhibits rac1 or CDC42Hsbinding to GST-PAKette, and preferably has an IC₅₀ (concentration atwhich 50% of maximal inhibition occurs) in the range of less than 1 μMand more preferably less than 1 nM. Most preferably an agent willexhibit selectivity in inhibition of binding by not substantiallyinhibiting other p21 protein:protein kinase pairs, preferably with atleast a two-fold, more preferably at least a three-fold, and even morepreferably at least a ten-fold selectivity.

Example 15

Screening for Compounds that Inhibit hPAK65 Kinase Activity

Compounds can be evaluated in an in vitro assay format for the abilityto inhibit human PAK65 kinase activity for the substrate MBP. Thegeneral assay formats are as provided in the Examples above. Anotherassay can be adapted (MacDonald et al. (1993) Mol. Cell. Biol. 13: 6615)as follows: purified human PAK65 protein and purified MBP are combinedin a reaction vessel (e.g., a microtiter plate well) under bufferedaqueous conditions in the presence of γ-³³ P-ATP and incubated at 32°C.; kinase catalyzed incorporation of ³³ P into protein is detected asradioactive counts retained on phosphocellulose paper after rinsing toremove unbound (i.e., non-protein) components, including γ-³³ P-ATP.Test agents are included in parallel reactions and evaluated for theirability to inhibit hPAK65-dependent incorporation of ³³ P into proteinretained on phosphocellulose. In an alternate embodiment, γ-³² P-ATP canbe used in place of γ-³³ P-ATP. In a second alternate embodiment, apeptide, such as CQQFGFGRRASDDG may be used in place of MBP (Kolch etal. (1993) Nature 364: 249).

A candidate agent is one which significantly inhibits hPAK65 kinaseactivity, and preferably has an IC₅₀ (concentration at which 50% ofmaximal inhibition occurs) in the range of approximately 3×10⁻⁸ M. Mostpreferably an agent will exhibit selectivity in inhibition of hPAK65 bynot substantially inhibiting protein kinase C (PKC) activity atconcentrations up to 1×10⁻⁵ M. More preferably, an agent should neithersignificantly inhibit rasGAP activity by 1×10⁻⁵ M agent under standardassay conditions for those activities.

Example 16

Inhibition of Human Tumor Growth in SCID Mouse

To evaluate the ability of a candidate agent to inhibit human tumorgrowth, human tumor cells were injected into SCID mice (severe combinedimmunodeficiency) to form palpable tumor masses. The effects of ancandidate agent in inhibiting tumor growth can be determined as follows.Approximately 1×10⁷ cells of the CCL 221 cell line (ATCC, Rockville,Md.), a human ras-dependent colon adenocarcinoma cell line, is suspendedin 100 μl DMEM and injected subcutaneously into SCID mice, such that twotumors per mouse are formed. SCID mice receive CCL 221 cells and thetumors are grown for 7 days without treatment; on the 7th day (Day 0)tumor maximal diameters and animal weights are recorded. On Day 0, themean tumor size for the mice is determined. On Day 1 (eight daysfollowing tumor cell injection), treatment of the mice with candidateagent or vehicle alone was begun. One group of the mice (controls) wereinjected intraperitoneally with 0.2 ml of vehicle and a second group ofmice received agent by intraperitoneal injection. Various doses of agentcan be tested in separate groups of mice. On Day 7 and Day 14, animalweight and maximal tumor diameter is measured. Average maximal tumorsize for each group on Day 0, Day 7, and Day 14 are compared Day 14, onehigh dose animal was followed for an additional to determined whetherthe agent produces a dose-dependent inhibition of tumor growth. Toxicityeffects can be examined by tracking mice weight and by harvesting lungs,livers, and spleens of the animals for histologically staining.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2248 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: hPAK65                                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 391..1908                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AGGGGAGGAC ACACTTCTGG CAAACGTTTC TCAAATCTGC TTCATCCAAT GT -             #GAAGTTCA     60                                                                 - - TCTTGCAGCA TTTACTATGC ACAACAGAGT AACTATCGGG TCCTGTGGAC AG -            #CTCACCTA    120                                                                 - - GTGGCAATGG CTCCAGGCTC CCGGACATCC CGTCTCCTGG CTTTTGCCTG CT -            #CTGCCTGC    180                                                                 - - CCTGGCTTCA AGAGGCTGGT GCGTCCAAAC CGTTCCGTTA TCCAGGCTTT TT -            #GACCACGC    240                                                                 - - TATGCTCCAA GCCCATGCGC GCACCAGCTG GCCATTGACA CCTACCAGGA GT -            #TTGAAGAA    300                                                                 - - ACCTATATCC CAAAGGACCA GAAGTATTCA TTCCTGCATG ACTCCCAGAC CT -            #CCTTCTGC    360                                                                 - - TTCTCAGACT CTATTCCGAC ACCCTCCAAC ATG GAG GAA ACG CA - #A CAG AAA       TCC     414                                                                                       - #               Met Glu - #Glu Thr Gln Gln Lys Ser                         - #                 1 - #              5                     - - AAT CTA GAG CTG CTG TCA GCC AAT CAC AGT TT - #G AAA CCT TTG CCC TCT          462                                                                       Asn Leu Glu Leu Leu Ser Ala Asn His Ser Le - #u Lys Pro Leu Pro Ser                10             - #     15             - #     20                          - - GTT CCA GAA GAG AAA AAG CCC AGG CAT AAA AT - #C ATC TCC ATA TTC TCA          510                                                                       Val Pro Glu Glu Lys Lys Pro Arg His Lys Il - #e Ile Ser Ile Phe Ser            25                 - # 30                 - # 35                 - # 40       - - GGC ACA GAG AAA GGA AGT AAA AAG AAA GAA AA - #G GAA CGG CCA GAA ATT          558                                                                       Gly Thr Glu Lys Gly Ser Lys Lys Lys Glu Ly - #s Glu Arg Pro Glu Ile                            45 - #                 50 - #                 55              - - TCT CCT CCA TCT GAT TTT GAG CAC ACC ATC CA - #T GTT GGC TTT GAT ACT          606                                                                       Ser Pro Pro Ser Asp Phe Glu His Thr Ile Hi - #s Val Gly Phe Asp Thr                        60     - #             65     - #             70                  - - GTT ACT GGA GAA TTC ACT GGC ATG CCA GAA CA - #G TGG GCT CGA TTA CTA          654                                                                       Val Thr Gly Glu Phe Thr Gly Met Pro Glu Gl - #n Trp Ala Arg Leu Leu                    75         - #         80         - #         85                      - - CAG ACC TCC AAT ATC ACC AAA CTA GAG CAA AA - #G AAG AAT CCT CAG GCT          702                                                                       Gln Thr Ser Asn Ile Thr Lys Leu Glu Gln Ly - #s Lys Asn Pro Gln Ala                90             - #     95             - #    100                          - - GTG CTG GAT GTC CTA AAG TTC TAC GAC TCC AA - #C ACA GTG AAG CAG AAA          750                                                                       Val Leu Asp Val Leu Lys Phe Tyr Asp Ser As - #n Thr Val Lys Gln Lys           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - TAT CTG AGC TTT ACT CCT CCT GAG AAA GAT GG - #C TTT CCT TCT GGA        ACA      798                                                                    Tyr Leu Ser Phe Thr Pro Pro Glu Lys Asp Gl - #y Phe Pro Ser Gly Thr                          125  - #               130  - #               135              - - CCA GCA CTG AAT GCC AAG GGA ACA GAA GCA CC - #C GCA GTA GTG ACA GAG          846                                                                       Pro Ala Leu Asn Ala Lys Gly Thr Glu Ala Pr - #o Ala Val Val Thr Glu                       140      - #           145      - #           150                  - - GAG GAG GAT GAT GAT GAA GAG ACT GCT CCT CC - #C GTT ATT GCC CCG CGA          894                                                                       Glu Glu Asp Asp Asp Glu Glu Thr Ala Pro Pr - #o Val Ile Ala Pro Arg                   155          - #       160          - #       165                      - - CCG GAT CAT ACA AAA TCA ATT TAC ACA CGG TC - #T GTA ATT GAC CCT GTT          942                                                                       Pro Asp His Thr Lys Ser Ile Tyr Thr Arg Se - #r Val Ile Asp Pro Val               170              - #   175              - #   180                          - - CCT GCA CCA GTT GGT GAT TCA CAT GTT GAT GG - #T GCT GCC AAG TCT TTA          990                                                                       Pro Ala Pro Val Gly Asp Ser His Val Asp Gl - #y Ala Ala Lys Ser Leu           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - GAC AAA CAG AAA AAG AAG ACT AAG ATG ACA GA - #T GAA GAG ATT ATG        GAG     1038                                                                    Asp Lys Gln Lys Lys Lys Thr Lys Met Thr As - #p Glu Glu Ile Met Glu                          205  - #               210  - #               215              - - AAA TTA AGA ACT ATC GTG AGC ATA GGT GAC CC - #T AAG AAA AAA TAT ACA         1086                                                                       Lys Leu Arg Thr Ile Val Ser Ile Gly Asp Pr - #o Lys Lys Lys Tyr Thr                       220      - #           225      - #           230                  - - AGA TAT GAA AAA ATT GGA CAA GGG GCT TCT GG - #T ACA GTT TTC ACT GCT         1134                                                                       Arg Tyr Glu Lys Ile Gly Gln Gly Ala Ser Gl - #y Thr Val Phe Thr Ala                   235          - #       240          - #       245                      - - ACT GAC GTT GCA CTG GGA CAG GAG GTT GCT AT - #C AAA CAA ATT AAT TTA         1182                                                                       Thr Asp Val Ala Leu Gly Gln Glu Val Ala Il - #e Lys Gln Ile Asn Leu               250              - #   255              - #   260                          - - CAG AAA CAG CCA AAG AAG GAA CTG ATC ATT AA - #C GAG ATT CTG GTG ATG         1230                                                                       Gln Lys Gln Pro Lys Lys Glu Leu Ile Ile As - #n Glu Ile Leu Val Met           265                 2 - #70                 2 - #75                 2 -      #80                                                                              - - AAA GAA TTG AAA AAT CCC AAC ATC GTT AAC TT - #T TTG GAC AGT TAC        CTG     1278                                                                    Lys Glu Leu Lys Asn Pro Asn Ile Val Asn Ph - #e Leu Asp Ser Tyr Leu                          285  - #               290  - #               295              - - GTA GGA GAT GAA TTG TTT GTG GTC ATG GAA TA - #C CTT GCT GGG AGG TCA         1326                                                                       Val Gly Asp Glu Leu Phe Val Val Met Glu Ty - #r Leu Ala Gly Arg Ser                       300      - #           305      - #           310                  - - CTC ACT GAT GTG GTA ACA GAA ACG TGC ATG GA - #T GAA GCA CAG ATT GCT         1374                                                                       Leu Thr Asp Val Val Thr Glu Thr Cys Met As - #p Glu Ala Gln Ile Ala                   315          - #       320          - #       325                      - - GCT GTA TGC AGA GAG TGT TTA CAG GCA TTG GA - #G TTT TTA CAT GCT AAT         1422                                                                       Ala Val Cys Arg Glu Cys Leu Gln Ala Leu Gl - #u Phe Leu His Ala Asn               330              - #   335              - #   340                          - - CAA GTG ATC CAC AGA GAC ATC AAA AGT GAC AA - #T GTA CTT TTG GGA ATG         1470                                                                       Gln Val Ile His Arg Asp Ile Lys Ser Asp As - #n Val Leu Leu Gly Met           345                 3 - #50                 3 - #55                 3 -      #60                                                                              - - GAA GGA TCT GTT AAG CTC ACT GAC TTT GGT TT - #C TGT GCC CAG ATC        ACC     1518                                                                    Glu Gly Ser Val Lys Leu Thr Asp Phe Gly Ph - #e Cys Ala Gln Ile Thr                          365  - #               370  - #               375              - - CCT GAG CAG AGC AAA CGC AGT ACC ATG GTC GG - #A ACG CCA TAC TGG ATG         1566                                                                       Pro Glu Gln Ser Lys Arg Ser Thr Met Val Gl - #y Thr Pro Tyr Trp Met                       380      - #           385      - #           390                  - - GCA CCA GAG GTG GTT ACA CGG AAA GCT TAT GG - #C CCT AAA GTC GAC ATA         1614                                                                       Ala Pro Glu Val Val Thr Arg Lys Ala Tyr Gl - #y Pro Lys Val Asp Ile                   395          - #       400          - #       405                      - - TGG TCT CTG GGT ATC ATG GCT ATT GAG ATG GT - #A GAA GGA GAG CCT CCA         1662                                                                       Trp Ser Leu Gly Ile Met Ala Ile Glu Met Va - #l Glu Gly Glu Pro Pro               410              - #   415              - #   420                          - - TAC CTC AAT GAA AAT CCC CTT AGG GCC TTG TA - #C CTA ATA GCA ACT AAT         1710                                                                       Tyr Leu Asn Glu Asn Pro Leu Arg Ala Leu Ty - #r Leu Ile Ala Thr Asn           425                 4 - #30                 4 - #35                 4 -      #40                                                                              - - GGA ACC CCA GAA CTT CAG AAT CCA GAG AAA CT - #T TCC CCA ATA TTT        CGG     1758                                                                    Gly Thr Pro Glu Leu Gln Asn Pro Glu Lys Le - #u Ser Pro Ile Phe Arg                          445  - #               450  - #               455              - - GAT TTC TTA AAT CGA TGT TTG GAA ATG GAT GT - #G GAA AAA AGG GGT TCA         1806                                                                       Asp Phe Leu Asn Arg Cys Leu Glu Met Asp Va - #l Glu Lys Arg Gly Ser                       460      - #           465      - #           470                  - - GCC AAA GAA TTA TTA CAG CAT CCT TTC CTG AA - #A CTG GCC AAA CCG TTA         1854                                                                       Ala Lys Glu Leu Leu Gln His Pro Phe Leu Ly - #s Leu Ala Lys Pro Leu                   475          - #       480          - #       485                      - - TCT AGC TTG ACA CCA CTG ATC ATG GCA GCT AA - #A GAA GCA ATG AAG AGT         1902                                                                       Ser Ser Leu Thr Pro Leu Ile Met Ala Ala Ly - #s Glu Ala Met Lys Ser               490              - #   495              - #   500                          - - AAC CGT TAACATCACT GCTGTGGCCT CATACTCTTT TTTCCATTTT CT - #ACAAGAAG          1958                                                                       Asn Arg                                                                       505                                                                            - - CCTTTTAGTA TATGAAAATT ATTACTCTTT TTGGGGTTTA AAGAAATGGT CT -             #GCATAACC   2018                                                                 - - TGAATGAAAG AAGCAAATGA CTATTCTCTG AAGACAACCA AGAGAAAATT GC -            #AAAAAGAC   2078                                                                 - - AAGTATGACT TTTATATGAA CCCCTTCTTT AGGGTCCAGA AGGAATTGTG GA -            #CTGAATCA   2138                                                                 - - CTAGCCTTAG GTCTTTCAGC AAACAGCCTA TCAGGGCCAT TTATCATGTG TG -            #AGATTTGC   2198                                                                 - - ATTTTACTTT GCTGACTTTG TTGTAATAGA TCCCATTCAT TGTCCCCTTT  - #                2248                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 506 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Glu Glu Thr Gln Gln Lys Ser Asn Leu Gl - #u Leu Leu Ser Ala Asn        1               5 - #                 10 - #                 15              - - His Ser Leu Lys Pro Leu Pro Ser Val Pro Gl - #u Glu Lys Lys Pro Arg                   20     - #             25     - #             30                  - - His Lys Ile Ile Ser Ile Phe Ser Gly Thr Gl - #u Lys Gly Ser Lys Lys               35         - #         40         - #         45                      - - Lys Glu Lys Glu Arg Pro Glu Ile Ser Pro Pr - #o Ser Asp Phe Glu His           50             - #     55             - #     60                          - - Thr Ile His Val Gly Phe Asp Thr Val Thr Gl - #y Glu Phe Thr Gly Met       65                 - # 70                 - # 75                 - # 80       - - Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Se - #r Asn Ile Thr Lys Leu                       85 - #                 90 - #                 95              - - Glu Gln Lys Lys Asn Pro Gln Ala Val Leu As - #p Val Leu Lys Phe Tyr                  100      - #           105      - #           110                  - - Asp Ser Asn Thr Val Lys Gln Lys Tyr Leu Se - #r Phe Thr Pro Pro Glu              115          - #       120          - #       125                      - - Lys Asp Gly Phe Pro Ser Gly Thr Pro Ala Le - #u Asn Ala Lys Gly Thr          130              - #   135              - #   140                          - - Glu Ala Pro Ala Val Val Thr Glu Glu Glu As - #p Asp Asp Glu Glu Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro Pro Val Ile Ala Pro Arg Pro Asp Hi - #s Thr Lys Ser Ile        Tyr                                                                                             165  - #               170  - #               175             - - Thr Arg Ser Val Ile Asp Pro Val Pro Ala Pr - #o Val Gly Asp Ser His                  180      - #           185      - #           190                  - - Val Asp Gly Ala Ala Lys Ser Leu Asp Lys Gl - #n Lys Lys Lys Thr Lys              195          - #       200          - #       205                      - - Met Thr Asp Glu Glu Ile Met Glu Lys Leu Ar - #g Thr Ile Val Ser Ile          210              - #   215              - #   220                          - - Gly Asp Pro Lys Lys Lys Tyr Thr Arg Tyr Gl - #u Lys Ile Gly Gln Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Ser Gly Thr Val Phe Thr Ala Thr Asp Va - #l Ala Leu Gly Gln        Glu                                                                                             245  - #               250  - #               255             - - Val Ala Ile Lys Gln Ile Asn Leu Gln Lys Gl - #n Pro Lys Lys Glu Leu                  260      - #           265      - #           270                  - - Ile Ile Asn Glu Ile Leu Val Met Lys Glu Le - #u Lys Asn Pro Asn Ile              275          - #       280          - #       285                      - - Val Asn Phe Leu Asp Ser Tyr Leu Val Gly As - #p Glu Leu Phe Val Val          290              - #   295              - #   300                          - - Met Glu Tyr Leu Ala Gly Arg Ser Leu Thr As - #p Val Val Thr Glu Thr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Cys Met Asp Glu Ala Gln Ile Ala Ala Val Cy - #s Arg Glu Cys Leu        Gln                                                                                             325  - #               330  - #               335             - - Ala Leu Glu Phe Leu His Ala Asn Gln Val Il - #e His Arg Asp Ile Lys                  340      - #           345      - #           350                  - - Ser Asp Asn Val Leu Leu Gly Met Glu Gly Se - #r Val Lys Leu Thr Asp              355          - #       360          - #       365                      - - Phe Gly Phe Cys Ala Gln Ile Thr Pro Glu Gl - #n Ser Lys Arg Ser Thr          370              - #   375              - #   380                          - - Met Val Gly Thr Pro Tyr Trp Met Ala Pro Gl - #u Val Val Thr Arg Lys      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ala Tyr Gly Pro Lys Val Asp Ile Trp Ser Le - #u Gly Ile Met Ala        Ile                                                                                             405  - #               410  - #               415             - - Glu Met Val Glu Gly Glu Pro Pro Tyr Leu As - #n Glu Asn Pro Leu Arg                  420      - #           425      - #           430                  - - Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pr - #o Glu Leu Gln Asn Pro              435          - #       440          - #       445                      - - Glu Lys Leu Ser Pro Ile Phe Arg Asp Phe Le - #u Asn Arg Cys Leu Glu          450              - #   455              - #   460                          - - Met Asp Val Glu Lys Arg Gly Ser Ala Lys Gl - #u Leu Leu Gln His Pro      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Phe Leu Lys Leu Ala Lys Pro Leu Ser Ser Le - #u Thr Pro Leu Ile        Met                                                                                             485  - #               490  - #               495             - - Ala Ala Lys Glu Ala Met Lys Ser Asn Arg                                              500      - #           505                                       __________________________________________________________________________

What is claimed is:
 1. A method for identifying a compound whichinhibits hPAK65 kinase activity, said method comprising:combining in areaction mixture a compound, a polypeptide comprising hPAK65 or afragment thereof having serine kinase activity, hPAK65 kinase substrate,and labeled ATP; and determining the extent to which the compoundinhibits phosphorylation of hPAK substrate as compared to a controlreaction lacking the compound.
 2. The method of claim 1, wherein saidinhibition of hPAK65 kinase activity is contingent upon activation of asignal transduction pathway.
 3. The method of claim 1, wherein saidcompound is selected from the group consisting of an antineoplasticagent, a cytotoxic agent, an inhibitor of neoplastic transformation, andan inhibitor of cell proliferation.
 4. The method of claim 1, whereinsaid polypeptide is a constitutively active hPAK65.
 5. The method ofclaim 4, wherein said polypeptide is selected from the group consistingof:(a) a truncation of hPAK65 missing the regulatory domain thatinhibits kinase activity, (b) an hPAK65 polypeptide having a glutamicacid substitution for serine 380, (c) an hPAK65 polypeptide having anaspartic acid substitution for serine 380, (d) an hPAK65 polypeptidehaving a glutamic acid substitution for serine 383, (e) an hPAK65polypeptide having an aspartic acid substitution for serine 383, (f) anhPAK65 polypeptide having a glutamic acid substitution for threonine384, (g) an hPAK65 polypeptide having an aspartic acid substitution forthreonine 384, (h) an hPAK65 polypeptide having C-terminal mutationswhere amino acids 489-491 are replaced non-conservatively, (i) an hPAK65polypeptide having C-terminal mutations where amino acids 489-491 aredeleted, and (j) an hPAK65 polypeptide having C-terminal mutations whereamino acids 482-506 are deleted.
 6. The method of claim 5, wherein saidpolypeptide is (j) and further comprises a myc-epitope tag.
 7. Themethod of claim 1, wherein said substrate is myelin basic protein (MBP).8. The method of claim 1, wherein said ATP is radiolabeled.
 9. A kit foridentifying agents which inhibit hPAK65 kinase activity, comprising apolypeptide containing hPAK65 or a fragment thereof having serine kinaseactivity and hPAK65 kinase substrate.
 10. The kit of claim 9, whereinsaid polypeptide is a constitutively activated hPAK65 or fragmentthereof with constitutive activity.
 11. The kit of claim 10, whereinsaid hPAK65 is selected from the group consisting of:(a) theautophosphorylated form, (b) an analog of hPAK65 having at least one ofan autophosphorylation-target serine or threonine replaced by an aminoacid that mimics phosphoserine, (c) an N-terminal truncated form ofhPAK65 in which the hPAK65 regulatory domain responsible for inhibitingserine kinase activity is deleted. (d) an hPAK65 polypeptide havingC-terminal mutations where amino acids 489-491 are replacednon-conservatively, (e) an hPAK65 polypeptide having C-terminalmutations where amino acids 489-491 are deleted, and (f) an hPAK65polypeptide having C-terminal mutations where amino acids 482-506 aredeleted.
 12. The kit of claim 11, wherein said amino acid that mimicsphosphoserine is Glu or Asp.
 13. The kit of claim 9, wherein saidsubstrate is myelin basic protein (MBP).
 14. The kit of claim 11,wherein said serine at position 381 is replaced by Glu or Asp.
 15. Thekit of claim 11, wherein said serine at position 383 is replaced by Gluor Asp.
 16. The kit of claim 11, wherein said threonine at position 384is replaced by Glu or Asp.
 17. The kit of claim 11, wherein saidpolypeptide is (f) and further comprises a myc-epitope tag.